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Study tests whether stem cells heal arthritis in large dogs – Los Angeles Times

About a year ago, Cheryl Timmons was worried her dog Baxter would soon need to retire from being a therapy dog due to arthritis in his hips.

The 99-pound German shepherds physical health was wearing down after years of bringing joy to childrens hospitals, senior homes and courtrooms, where he served as the first and only service dog providing comfort to child trafficking victims in Orange County.

Timmons, who rescued Baxter from the streets of San Bernardino, worried that she may even have to put him down.

To combat the worsening arthritis, Timmons took him to therapy sessions. A GoFundMe campaign to help pay for the therapy reached a goal of $4,500.

But the arthritis was still taking hold, affecting how Baxter functioned during long workdays.

Then in late August, he was given stem cell injections as part of a new study at the Anaheim Hills Pet Clinic. The effort, headed by San Diego-based Animal Cell Therapies, is testing whether stem cells can help alleviate arthritis in dogs weighing 70 pounds or more.

Baxter, now 11 years old, has been feeling better since he received his injection.

His arthritis is greatly improved, Timmons said. I swear by the stem cell treatment. It has made such a huge difference.

Everybody in the court would notice that he wasnt having a good day. Now hes looking great again. Hes running through the courtroom. He is one happy boy.

Baxter was one of about 10 dogs that was tested at the Anaheim clinic. Animal Cell Therapies is conducting the testing at a dozen clinics throughout the country.

There are about 35 dogs currently enrolled in the study. Researchers are hoping to test between 60 and 80 dogs.

Kathy Petrucci, chief executive of Animal Cell Therapies, said its too early to tell whether the treatment is successful in treating arthritis in large dogs, but the early results are promising.

The company conducted a similar study a year ago, which showed benefits for arthritis in dogs under 70 pounds. However, the results were mixed for bigger dogs.

Petrucci said they increased the dosage for the current study.

We dont know every single mechanism that is involved ... it helps decrease inflammation in the joints, Petrucci said of the treatment. We think that the cells secrete a lot of positive beneficial growth factors that help decrease inflammation, help make the environment a better, more friendly place for more normal cells to come in and help repair the joints.

Whatever the cause, Timmons just hopes the treatment allows Baxter to keep doing what he does best.

With the stem cells, he acts like hes invincible, Timmons said, laughing. I really hope he is.

To enroll in the study, visit dogstemcellstudy.com.

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Study tests whether stem cells heal arthritis in large dogs - Los Angeles Times

Stem cells and the heartthe road ahead – Science Magazine

Heart disease is the primary cause of death worldwide, principally because the heart has minimal ability to regenerate muscle tissue. Myocardial infarction (heart attack) caused by coronary artery disease leads to heart muscle loss and replacement with scar tissue, and the heart's pumping ability is permanently reduced. Breakthroughs in stem cell biology in the 1990s and 2000s led to the hypothesis that heart muscle cells (cardiomyocytes) could be regenerated by transplanting stem cells or their derivatives. It has been 18 years since the first clinical trials of stem cell therapy for heart repair were initiated (1), mostly using adult cells. Although cell therapy is feasible and largely safe, randomized, controlled trials in patients show little consistent benefit from any of the treatments with adult-derived cells (2). In the meantime, pluripotent stem cells have produced bona fide heart muscle regeneration in animal studies and are emerging as leading candidates for human heart regeneration.

In retrospect, the lack of efficacy in these adult cell trials might have been predicted. The most common cell type delivered has been bone marrow mononuclear cells, but other transplanted cell types include bone marrow mesenchymal stromal cells and skeletal muscle myoblasts, and a few studies have used putative progenitors isolated from the adult heart itself. Although each of these adult cell types was originally postulated to differentiate directly into cardiomyocytes, none of them actually do. Indeed, with the exception of skeletal muscle myoblasts, none of these cell types survive more than a few days in the injured heart (see the figure). Unfortunately, the studies using bone marrow and adult resident cardiac progenitor cells were based on a large body of fraudulent work (3), which has led to the retraction of >30 publications. This has left clinical investigators wondering whether their trials should continue, given the lack of scientific foundation and the low but measurable risk of bleeding, stroke, and infection.

Additionally, investigators have struggled to explain the beneficial effects of adult cell therapy in preclinical animal models. Because none of these injected cell types survive and engraft in meaningful numbers or directly generate new myocardium, the mechanism has always been somewhat mysterious. Most research has focused on paracrine-mediated activation of endogenous repair mechanisms or preventing additional death of cardiomyocytes. Multiple protein factors, exosomes (small extracellular vesicles), and microRNAs have been proposed as the paracrine effectors, and an acute immunomodulatory effect has recently been suggested to underlie the benefits of adult cell therapy (4). Regardless, if cell engraftment or survival is not required, the durability of the therapy and need for actual cells versus their paracrine effectors is unclear.

Of particular importance to clinical translation is whether cell therapy is additive to optimal medical therapy. This remains unclear because almost all preclinical studies do not use standard medical treatment for myocardial infarction. Given the uncertainties about efficacy and concerns over the veracity of much of the underlying data, whether agencies should continue funding clinical trials using adult cells to treat heart disease should be assessed. Perhaps it is time for proponents of adult cardiac cell therapy to reconsider the approach.

Pluripotent stem cells (PSCs) include embryonic stem cells (ESCs) and their reprogrammed cousins, induced pluripotent stem cells (iPSCs). In contrast to adult cells, PSCs can divide indefinitely and differentiate into virtually every cell type in the human body, including cardiomyocytes. These remarkable attributes also make ESCs and iPSCs more challenging to control. Through painstaking development, cell expansion and differentiation protocols have advanced such that batches of 1 billion to 10 billion pharmaceutical-grade cardiomyocytes, at >90% purity, can be generated.

Preclinical studies indicate that PSC-cardiomyocytes can remuscularize infarcted regions of the heart (see the figure). The new myocardium persists for at least 3 months (the longest time studied), and physiological studies indicate that it beats in synchrony with host myocardium. The new myocardium results in substantial improvement in cardiac function in multiple animal models, including nonhuman primates (5). Although the mechanism of action is still under study, there is evidence that these cells directly support the heart's pumping function, in addition to providing paracrine factors. These findings are in line with the original hope for stem cell therapyto regenerate lost tissue and restore organ function. Additional effects, such as mechanically buttressing the injured heart wall, may also contribute.

Breakthroughs in cancer immunotherapy have led to the adoption of cell therapies using patient-derived (autologous) T cells that are genetically modified to express chimeric antigen receptors (CARs) that recognize cancer cell antigens. CAR T cells are the first U.S. Food and Drug Administration (FDA)approved, gene-modified cellular pharmaceutical (6). The clinical and commercial success of autologous CAR T cell transplant to treat B cell malignancies has opened doors for other complex cell therapies, including PSC derivatives. There is now a regulatory path to the clinic, private-sector funding is attracted to this field, and clinical investigators in other areas are encouraged to embrace this technology. Indeed, the first transplants of human ESC-derived cardiac progenitors, surgically delivered as a patch onto the heart's surface, have been carried out (7). In the coming years, multiple attempts to use PSC-derived cardiomyocytes to repair the human heart are likely.

What might the first human trials look like? These studies will probably employ an allogeneic (non-self), off-the-shelf, cryopreserved cell product. Although the discovery of iPSCs raised hopes for widespread use of autologous stem cell therapies, the current technology and regulatory requirements likely make this approach too costly for something as common as heart disease, although this could change as technology and regulations evolve. Given that it would take at least 6 months to generate a therapeutic dose of iPSC-derived cardiomyocytes, such cells could only be applied to patients whose infarcts are in the chronic phase where scarring (fibrosis) and ventricular remodeling are complete. Preclinical data indicate that chronic infarcts benefit less from cardiomyocyte transplantation than do those with active wound-healing processes.

Adult cells from bone marrow or the adult heart secrete beneficial paracrine factors but do not engraft in the infarcted heart. Pluripotent stem cells give rise to cardiomyocytes that engraft long term in animal models, beat in synchrony with the heart, and secrete beneficial paracrine factors. Long-term cardiomyocyte engraftment partially regenerates injured heart, which is hypothesized to bring clinical benefits.

The need for allogeneic cells raises the question of how to prevent immune rejection, both from innate immune responses in the acute phase of transplantation or from adaptive immune responses that develop more slowly through the detection of non-self antigens presented by major histocompatibility complexes (MHCs). A current strategy is the collection of iPSCs from patients who have homozygous MHC loci, which results in exponentially more MHC matches with the general population. However, studies in macaque monkeys suggest that MHC matching will be insufficient. In a macaque model of brain injury, immunosuppression was required to prevent rejection of MHC-matched iPSC-derived neurons (8). Similarly, MHC matching reduced the immunogenicity of iPSC-derived cardiomyocytes transplanted subcutaneously or into the hearts of rhesus macaques, but immunosuppressive drugs were still required to prevent rejection (9).

Numerous immune gene editing approaches have been proposed to circumvent rejection, including preventing MHC class I and II molecule expression, overexpressing immunomodulatory cell-surface factors, such CD47 and human leukocyte antigen E (HLA-E) and HLA-G (two human MHC molecules that promote maternal-fetal immune tolerance), or engineering cells to produce immunosuppressants such as programmed cell death ligand 1 (PDL1) and cytotoxic T lymphocyteassociated antigen 4 (CTLA4) (10). These approaches singly or in combination seem to reduce adaptive immune responses in vitro and in mouse models. Overexpressing HLA-G or CD47 also blunts the innate natural killer cellmediated response that results from deleting MHC class I genes (11). However, these manipulations are not without theoretical risks. It could be difficult to clear viral infections from an immunostealthy patch of tissue, and possible tumors resulting from engraftment of PSCs might be difficult to clear immunologically.

Ventricular arrhythmias have emerged as the major toxicity of cardiomyocyte cell therapy. Initial studies in small animals showed no arrhythmic complications (probably because their heart rates are too fast), but in large animals with human-like heart rates, arrhythmias were consistently observed (5, 12). Stereotypically, these arrhythmias arise a few days after transplantation, peak within a few weeks, and subside after 4 to 6 weeks. The arrhythmias were well tolerated in macaques (5) but were lethal in a subset of pigs (12). Electrophysiological studies indicate that these arrhythmias originate in graft regions from a source that behaves like an ectopic pacemaker. Understanding the mechanism of these arrhythmias and developing solutions are major areas of research. There is particular interest in the hypothesis that the immaturity of PSC-cardiomyocytes contributes to these arrhythmias, and that their maturation in situ caused arrhythmias to subside.

A successful therapy for heart regeneration also requires understanding the host side of the equation. PSC-derived cardiomyocytes engraft despite transplantation into injured myocardium that is ischemic with poor blood flow. Although vessels eventually grow in from the host tissue, normal perfusion is not restored. Achieving a robust arterial input will be key to restoring function, which may require cotransplanting other cell populations or tissue engineering approaches (13, 14). Most PSC-mediated cardiac cell therapy studies have been performed in the subacute window, equivalent to 2 to 4 weeks after myocardial infarction in humans. At this point, there has been insufficient time for a substantial fibrotic response. Fibrosis has multiple deleterious features, including mechanically stiffening the tissue and creating zones of electrical insulation that can cause arrhythmias. Extending this therapy to other clinical situations, such as chronic heart failure, will require additional approaches that address the preexisting fibrosis. Cell therapy may again provide an answer because CAR T cells targeted to cardiac fibroblasts reduced fibrosis (15).

Developing a human cardiomyocyte therapy for heart regeneration will push the limits of cell manufacturing. Each patient will likely require a dose of 1 billion to 10 billion cells. Given the widespread nature of ischemic heart disease, 105 to 106 patients a year are likely to need treatment, which translates to 1014 to 1016 cardiomyocytes per year. Growing cells at this scale will require introduction of next generation bioreactors, development of lower-cost media, construction of large-scale cryopreservation and banking systems, and establishment of a robust supply chain compatible with clinical-grade manufacturing practices.

Beyond PSC-cardiomyocytes, other promising approaches include reactivating cardiomyocyte division and reprogramming fibroblasts to form new cardiomyocytes. However, these approaches are at an earlier stage of development, and currently, PSC-derived cardiomyocyte therapy is the only approach that results in large and lasting new muscle grafts. The hurdles to this treatment are known, and likely addressable, thus multiple clinical trials are anticipated.

Acknowledgments: C.E.M. and W.R.M. are scientific founders of and equity holders in Sana Biotechnology. C.E.M. is an employee of Sana Biotechnology. W.R.M. is a consultant for Sana Biotechnology. C.E.M. and W.R.M. hold issued and pending patents in the field of stem cell and regenerative biology.

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Stem cells and the heartthe road ahead - Science Magazine

On the Trail of Cancer Stem Cells – Technology Networks

Two research teams from the Max Delbrck Center for Molecular Medicine and their collaborators have produced a detailed cell atlas of an entire salivary gland tumor in a mouse model, mapping individual cells throughout the tumor and its surrounding tissue. The "single cell" approach, recently described inNature Communications, has provided key insights about cellular composition changes through the earliest stages of cancer development.

A solid tumor is not, as many might assume, a lump of cells that are all the same. Rather it is mix of many different cell types, including a variety of stromal and immune cells besides the actual tumor cells.

"Conventional methods in molecular biology often consider a sample as a whole, which fails to recognize the complexity within it," said Dr. Samantha Praktiknjo, senior scientist and first author from MDC's Systems Biology of Gene Regulatory Elements Lab headed by Professor Nikolaus Rajewsky at the Berlin Institute for Medical Systems Biology (BIMSB). Developing a detailed understanding of the different cells within a tumor and how they interact could help identify more effective treatment strategies.Strength in numbers

The team used single-cell RNA sequencing technologies developed in theRajewsky laband novel epitope profiling to produce the cell atlas, and identified the cells that were specific to the tumor by leveraging the reproducibility and the large sample size of their data.

The latter was possible by using a mouse model, developed in MDC's Signal Transduction in Development and Cancer Lab headed by Professor Walter Birchmeier, which harbors designed mutations that induce a salivary gland squamous cell carcinoma. This system provides a consistent supply of genetically similar tumors to sequence from the earliest stages of development, which is nearly impossible with human patients.

"In a patient, the tumor is already developed and you cannot go back and rewind time and look at how it started," said Dr. Benedikt Obermayer, a co-first author now at the Berlin Institute of Health (BIH). "Here, we have a model that is so controlled, we can watch it happen." And Dr. Qionghua Zhu, the third first author and a former postdoc at theBirchmeier Lab, added: "To fight cancer effectively, we need to find the driver mutations. This method gives us clues about the evolution trajectories of a tumor."

Sequencing technologies have advanced so that it is now possible to quickly and affordably sequence the RNA inside single cells, one at a time, as well as the proteins on the surfaces of cells in the tissues. While other methods grind up the tissue and identify what genes and molecules are present in the mix, the single cell approach precisely identifies how many of each type of cell is present, and which genes and molecules are associated with which cell.

For this study, the researchers sequenced more than 26,000 individual salivary gland cells from mice with tumors and healthy mice. They used computational models to analyze the huge amount of data and identify each individual cell and sort them into groups - such as stromal cells, immune cells, saliva producing cells, cancer cells - based on the hundreds of genes expressed and molecules present.A surprise

The single cell approach revealed something that surprised the researchers: "When I saw the data, I thought, where is the tumor?" Obermayer said. The population of cancer stem cells in the tumor was extremely small - less than one percent of all profiled cells in the tissue. Due to their low abundance, investigation of these cells still heavily depends on assumptions about surface markers and is often performed in cell culture-based systems. Here, the authors were able to identify the cancer stem cells directly from the solid tumor samples with their single cell approach.

Furthermore, the team was able to predict the progression of the different cell types through different stages of tumor development. Their model suggests that the cancer stem cells emerge from cancerous basal cells, then develop into another subtype before ultimately becoming a population of cells similar to luminal cells, a cell type present in normal, healthy salivary glands.

This progression supports the idea that when something goes awry in the basal cells of this solid tumor model, they are triggered to turn into cancer stem cells, which can then become a different type of cell. "What I found fascinating was clearly seeing the order of signals and events, transitioning from the progenitor to the progeny populations of the cancer stem cells," Praktiknjo said.Next steps

Further research is required to verify that individual cells are transforming through these stages, and explore the cellular and molecular interactions driving tumor growth. The team anticipates the approach they've demonstrated here can be applied to other cancer types as well.

"To me the main conceptual insight is that we can apply ideas from single-cell based developmental biology to reconstruct molecular progression of tumorigenesis ," said Professor Nikolaus Rajewsky, who heads MDC's Systems Biology of Gene Regulatory Elements Lab and is the scientific director of the BIMSB.

Reference:Praktiknjo, et al. (2020) Tracing tumorigenesis in a solid tumor model at single-cell resolution, Nature Communications, DOI: 10.1038/s41467-020-14777-0.

This article has been republished from the following materials. Note: material may have been edited for length and content. For further information, please contact the cited source.

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On the Trail of Cancer Stem Cells - Technology Networks

Report: Austin Aries DDP Trying To Get AEW To Buy Into Stem Cell Treatment Venture – Fightful

Austin Aries was backstage at AEW Dynamite in Atlanta, GA last night and according to a new report, he was pitching a stem cell treatment venture.

According to Cassidy Haynes of Bodyslam.net, Aries and Diamond Dallas Page were at AEW Dynamite trying to get AEW to buy into a stem cell treatment venture that they are part of.

Wrestlers have taken to stem cell treatment in past few years with Rey Mysterio, Brian Cage, John Morrison, Rob Van Dam, and more undergoing stem cell treatment to help prolong their careers and deal with past injuries. Aries has mentioned BioXcellerator stem cell therapy on his social media in the past.

Recently, Aries has been hanging out with Page and taken to DDP Yoga. Page has a DDP Yoga studio based in Georgia and is a regular at AEW events due to his relationship with Cody Rhodes. Page has also been featured on AEW television in past months, including wrestling a match on the Bash at the Beach (Jan. 15) episode of AEW Dynamite.

Aries last wrestled for Major League Wrestling and is currently a free agent.

If you missed anything from last night's AEW Dynamite, you can check out Fightful's full report on the show by clicking here.

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Report: Austin Aries DDP Trying To Get AEW To Buy Into Stem Cell Treatment Venture - Fightful

Rare disease outlook 2020: three therapies set to make waves this year – pharmaceutical-technology.com

Understanding the genetic causes of rare diseases supports drug development. Credit: Shutterstock.

Developing drugs to treat rare diseases is fraught with challenges; these range from trying to recruit from tiny patient populations to fill much-need clinical trials to the complex reimbursement landscape for these innovative, and often bespoke, therapies. However, as scientists improve their understanding of the genetic causes of many rare conditions and regulators explore new reimbursement options, pharma companies and smaller biotech firms are increasingly being empowered to address more of these tricky indications.

In this context, could 2020 be a breakthrough year for patients with rare diseases? Here are three case studies of companies on the verge of having treatments for rare diseases approved Rocket and Fanconi anaemia, PTC Therapeutics and aromatic l-amino acid decarboxylase (AADC) deficiency and, finally, Amryt and epidermolysis bullosa.

Fanconi anaemia (FA) is a rare paediatric inherited diseasecharacterised by bone marrow failure and predisposition to cancer, in the words of Rocket Pharmas CEO Gaurav Shah. Caused by a mutation in the FANC genes, patients with Fanconi experience bone marrow failure as they are unable to create new blood cells.

The current standard of care for Fanconi is a stem cell transplant, but Shah explains the risks involved with these pioneering procedures.

While these transplants do prolong patients lives, the procedure is incredibly difficult and is associated with a high potential for graft-versus-host disease, he says. Stem cell transplants can also lead to an even higher risk of head and neck cancer risk for Fanconi patients; almost everyone with FA who undergoes this procedure dies in their 30s.

Rocket wants to change this situation with its lentiviral vector gene therapy, RP-L102. It is specifically for Fanconi-A, which Shah explains is the most common form of the disease. He adds that the therapy contains patient-derived haematopoietic stem cells that have been generally modified to contain a functional copy of FANCA gene, a mutation which causes Fanconi-A.

RP-L102 is currently in a global registrational Phase IIA study, which has been efficacious and safe in patients so far. The data demonstrate that a single dose of RP-L102 leads to both genetic and functional correction as measured by a progressive increase in corrected peripheral blood and bone marrow cells, says Shah. Most importantly, this treatment can be administered without a conditioning regimen [of chemotherapy and radiation]. [This] means we may be able to treat patients as a preventative measure before bone marrow failure occurs, like a vaccine, with a single dose administration early in life.

Based on these promising signals, RP-L102 has received all accelerated regulatory tools from the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA). The company is hoping to complete its biologics license applications and marketing authorisation applications (MAA) to the two regulators within the next few years.

To overcome challenges facing Rocket in the development of RP-L102, Shah explains the company worked to improve upon its own expertise in rare diseases by working with world-class research and development partners, as well immersing itself within patient communities to learn more about their treatment needs.

Slightly further along the drug approval journey is PTC Therapeutics AADC deficiency drug, PTC-AADC, for which the company recently submitted an MAA to the EMA. The company expects full EMA approval towards the end of 2020 and to treat the first patients either in the first or second quarter of 2021.

PTC acquired PTC-AADC, alongside other gene therapy assets, when it bought rare central nervous system-focused Agilis Biotherapeutics in July 2018, PTCs EMEA and Asia Pacific senior vice-president and general manager Adrian Haigh explains.

AADC deficiency is a rare condition caused by a mutation in the DDC gene, which leads to issues with the AADC enzyme and subsequent reductions in the production of dopamine. Children suffering with AADC deficiency fail to reach neurological and development milestones and have a high risk of death early in life. The only current approach to treating the condition is through dopamine agonists, which Haigh notes are largely ineffective.

The particular approach developed by Agilis, [which is] unlike other forms of gene therapy, involves delivering a very small dose of gene therapy directly into the affected, post-mitotic cells, Haigh says. The rationale is that once youve delivered the drug to post-mitotic cells, which are not dividing, it is going to stay there for a long time.

Other advantages include a reduced chance of significant immune reaction and since the dose is smaller, the treatment could overcome some of the manufacturing issues facing other gene therapies. PTC has decided to bring PTC-AADCs manufacturing in house so they are not reliant on third parties schedules and capacities.

PTCs MAA for its AADC deficiency gene therapy is based on two clinical trials of 26 patients in total. Haigh explains the company has mapped motor milestones, and he noted that in advisory boards with payers theyve been incredibly impressed by our videos showing children progressing from lying flat on their backs to walking around.

He notes that in this case, it is certainly not ethical to drill a hole in a patients head and inject a virus containing a placebo and instead PTC has successfully completed a single-arm trial by comparing with patients natural history. Regulators need to be open to novel clinical trial design, particularly in rare diseases where you have ethical problems, Haigh argues.

The company had to abandon a previous drug in development because they could not agree an economic and deliverable clinical trial design with the FDA.

One of the main challenges that faced PTC in the development of PTC-AADC was diagnosis. Haigh explains they found a lot of patients have been misdiagnosed with either cerebral palsy or epilepsy so the company launched a free genetic testing programme. This also allowed them to find patients to recruit into the trial and estimate the number of patients with AADC deficiency who might be able to benefit from this gene therapy.

Epidermolysis bullosa (EB) is a group of rare skin conditions caused by genetic mutations in the genes that encode for the proteins of the skin, particularly in collagen VII.

There are currently no approved treatments for this condition, EB charity DEBRAs UK branch director of research Caroline Collins notes the condition is managed by regular changing of dressings and the lancing of blisters.

EB is characterised by blisters and wounds on the skin; these wounds are extremely painful and can cover huge areas of the patients body, such as their whole back or entire legs. However, Collins explains these are not like the kinds of wounds you get with ulcers or burns, and they move continuously.

As well as making it incredibly challenging for patients to deal with these never-healing wounds, it also makes it difficult for drug developers to find and establish accepted clinical trial endpoints centred on wound healing. DEBRA is therefore advocating for natural history to be considered in clinical trial designs, Collins explains.

Despite these challenges, UK drug company Amryt is hoping to submit authorisation applications to the FDA and EMA by the end of 2021 for its EB drug, AP101. The company has repurposed the topical gel created for burns wounds to treat EB. It is made from a combination of an extract from the bark of the birch tree and pure sunflower oil, the companys chief medical officer Dr Mark Sumeray explains.

AP101 is currently being studied in a Phase III study Amryt claim this is the biggest global EB trial ever undertaken and has been granted rare paediatric disease designation from the FDA.

Although the current results are blinded, Sumeray explains a recent analysis by an independent data monitoring board found that the firm only needed to increase the number of patients slightly, suggesting that at this point in time, the data would have looked encouraging. Too small a patient population makes it hard for efficacy to be statistically significant.

Since Amryts AP101 may be the first drug approved for EB, Collins emphasises it is important that the company has productive conversations with regulators about the specific challenges of EB. This will help to set the ground for others to follow and further transform the lives of EB patients.

It is clear that Amryt is committed to EB because the company in-licensed a second EB candidate, a topical gene therapy called AP103 in 2018.

Sumeray explains: We have invested a lot of time and effort in the development, not only of the lead product, but also of relationships with physicians and scientists working in EB. If we can figure out how to successfully bring products to the market and have them reimbursed, then all of that knowledge can applied again.

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Rare disease outlook 2020: three therapies set to make waves this year - pharmaceutical-technology.com

Dutch startup Neuroplast raises 4M for its stem cell-based technology to treat patients with Spinal Cord Injury – Silicon Canals

Neuroplast is a company based in Maastricht (the Netherlands) developing autologous stem cell therapies for patients suffering from neurodegenerative diseases such as spinal cord injury (SCI), amyotrophic lateral sclerosis (ALS) and traumatic brain injury.

Recently, the company has raised 4 million from Dutch-based Brightlands Venture Partners and LIOF and from an existing shareholder and informal investor Lumana Invest BV.

CEO Johannes de Munter said:

The financing and support of the investors will enable us to perform multicenter clinical trials in the Netherlands, Denmark, Germany, and Spain and bring the product to market.

This Dutch startup will use the fund to perform a phase II/III clinical trial with the aim of obtaining conditional market approval for the treatment of patients suffering from Spinal Cord Injury.

Founded by physician Hans de Munter and neurologist Erik Wolters in 2014, Neuroplast has expanded with Juliette van den Dolder, who was appointed as COO and management team member.

In the case of SCI, isolating, manufacturing, and reinserting patients own cells, very promising preclinical outcomes have resulted in an Orphan Drug Designation from European regulatory authorities, allowing a fast-track procedure for the clinical trials. These trials are expected to start in March 2020.

Marcel Kloosterman Director at Brightlands Venture Partners:

Neuroplast combines breakthrough science with a solid management team. In a sizable market characterised by major unmet need, successful treatment of (accident caused) paralysed patients would make life so much easier for them and their families while lowering the burden and costs for the society.

Yearly, 24,500 people in Europe and the USA are diagnosed with Spinal Cord Injury, usually caused by accident. Its worth mentioning that for Europe and the US, the medical cost associated with Spinal Cord Injury is over 13 bn per year.

CEO Johannes de Munter adds:

Neuroplast is becoming an ATMP player in the region and wants to contribute to our beautiful eco-system.

Main image credits:Neuroplast

Stay tuned toSilicon Canalsfor more European technology news

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Dutch startup Neuroplast raises 4M for its stem cell-based technology to treat patients with Spinal Cord Injury - Silicon Canals

Animal Stem Cell Therapy Market 2020-2026: Product Types, by Applications, By Market Trends, Market Reserach Report – Keep Reading

Our latest research report entitle Global Animal Stem Cell Therapy Market provides comprehensive and deep insights into the market dynamics and growth of Global Animal Stem Cell Therapy Industry. Latest information on market risks, industry chain structure, Animal Stem Cell Therapy cost structure and opportunities are offered in this report. The entire industry is fragmented based on geographical regions, a wide range of applications and Global Animal Stem Cell Therapy Market types. The past, present and forecast market information will lead to investment feasibility by studying the crucial Global Animal Stem Cell Therapy Industry growth factors.

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VETSTEM BIOPHARMAMediVet BiologicJ-ARMCelavetMagellan Stem CellsU.S. Stem CellCells Power JapanANIMAL CELL THERAPIESAnimal Care StemCell Therapy SciencesVetCell TherapeuticsAnimacelAratana Therapeutics

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Europe Market (Germany, France, Italy, Russia and UK)

North America Market (Canada, USA and Mexico)

Latin America Market (Middle and Africa).

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Asia-Pacific Market (South-east Asia, China, India, Korea and Japan).

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Types Of Global Animal Stem Cell Therapy Market:

DogsHorsesOthers

Applications Of Global Animal Stem Cell Therapy Market:

Veterinary HospitalsResearch Organizations

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1 Market Overview

2 Global Animal Stem Cell Therapy Market Competition by Manufacturers

3 Global Animal Stem Cell Therapy Capacity, Production, Revenue (Value) by Region (2020-2026)

4 Global Animal Stem Cell Therapy Industry Supply (Production), Consumption, Export, Import by Region (2020-2026)

5 Global Animal Stem Cell Therapy Production, Revenue (Value), Price Trend by Type

6 Global Animal Stem Cell Therapy Market Analysis by Application

7 Global Animal Stem Cell Therapy Industry Manufacturers Profiles/Analysis

8. Animal Stem Cell Therapy Manufacturing Cost Analysis

9 Industrial Chain, Sourcing Strategy and Downstream Buyers

10 Marketing Strategy Analysis, Distributors/Traders

11 Market Effect Factors Analysis

12 Global Animal Stem Cell Therapy Market Forecast (2020-2026)

13 Research Findings and Conclusion

14 Appendix

Explore Full Report With Detailed TOC Here @ https://www.globalmarketers.biz/report/life-sciences/2018-global-animal-stem-cell-therapy-industry-research-report/118224 #table_of_contents

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Animal Stem Cell Therapy Market 2020-2026: Product Types, by Applications, By Market Trends, Market Reserach Report - Keep Reading

Global demand of Stem Cell Therapy Market will boom in coming years – Nyse Nasdaq Live

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The study included research on a variety of factors affecting the industry, including government policies, market conditions, competitive environments, historical data, current market trends, technological innovations, technological advances in future technologies and related industries, and market risks.Opportunities, market barriers and challenges.

Best Merchant Analysis is one of the key components and is very useful for each player to understand the focused scene in the market. The main major companies in the Stem Cell Therapy market report are: Osiris Therapeutics, Inc. Novartis AG, GlaxoSmithKline Plc., MEDIPOST Co., Ltd., Anterogen Co., Ltd. Pharmicell Co., Ltd. Holostem Terapie Avanzate S.r.l. JCR Pharmaceuticals Co., Ltd. NuVasive, Inc. RTI Surgical, Inc., and Fibrocell Science, Inc.

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Market Driver

Market Challenges

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Key Questions on the Stem Cell Therapy Market 20202026:

What will the market size be in 2026 and what will the growth rate be?

What are the main market trends?

What are the main factors driving this market?

What are the challenges to market growth?

Who are the main major suppliers in this market?

What are the market opportunities, market risks and market overview threats faced by major suppliers?

What are the Strengths and Weaknesses of the Major Suppliers?

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Global demand of Stem Cell Therapy Market will boom in coming years - Nyse Nasdaq Live

Combination Enfortumab Vedotin + Pembrolizumab Granted Breakthrough Therapy in Bladder Cancer – OncoZine

The U.S. Food and Drug Administration (FDA) has granted Breakthrough Therapy designation to enfortumab vedotin-ejfv (Padcev; Astellas Pharma and Seattle Genetics) in combination with Mercks (known as MSD outside the United States and Canada) anti-PD-1 therapy pembrolizumab (Keytruda) for the treatment of patients with unresectable locally advanced or metastatic urothelial cancer who are unable to receive cisplatin-based chemotherapy in the first-line setting.

It is estimated that approximately 81,000 people in the U.S. will be diagnosed with bladder cancer in 2020. [1] Urothelial cancer accounts for 90% of all bladder cancers and can also be found in the renal pelvis, ureter, and urethra. [2] Globally, approximately 549,000 people were diagnosed with bladder cancer in 2018, and there were approximately 200,000 deaths worldwide. [3]

The recommended first-line treatment for patients with advanced urothelial cancer is cisplatin-based chemotherapy. For patients who are unable to receive cisplatin, such as people with kidney impairment, a carboplatin-based regimen is recommended. However, fewer than half of patients respond to carboplatin-based regimens and outcomes are typically poorer compared to cisplatin-based regimens. [4]

Conditionally approvedEnfortumab vedotin-ejfv, a first-in-class antibody-drug conjugate (ADC) that is directed against Nectin-4, a protein located on the surface of cells and highly expressed in bladder cancer, was conditionally approved by the FDA in December 2019 based on the Accelerated Approval Program. [5][6]

Antibody-drug Conjugates or ADCs are highly targeted biopharmaceutical drugs that combine monoclonal antibodies specific to surface antigens present on particular tumor cells with highly potent anti-cancer agents linked via a chemical linker.

With seven approved drugs on the market, ADCs have become a powerful class of therapeutic agents in oncology and hematology.

Continued approval for enfortumab vedotin-ejfv in combination with pembrolizumab for the treatment of patients with advanced or metastatic urothelial cancer may be contingent upon verification and description of clinical benefit in confirmatory trials. [5]

The drug is indicated for the treatment of adult patients with locally advanced or metastatic urothelial cancer who have previously received a programmed death receptor-1 (PD-1) or programmed death-ligand 1 (PD-L1) inhibitor and a platinum-containing chemotherapy before (neoadjuvant) or after (adjuvant) surgery or in a locally advanced or metastatic setting.

Nonclinical data suggest the anticancer activity of enfortumab vedotin is due to its binding to Nectin-4 expressing cells followed by the internalization and release of the anti-tumor agent monomethyl auristatin E (MMAE) into the cell, which result in the cell not reproducing (cell cycle arrest) and in programmed cell death (apoptosis). [5]

Breakthrough therapyThe Breakthrough Therapy process is designed to expedite the development and review of drugs that are intended to treat a serious or life-threatening condition. The designation is based upon preliminary clinical evidence indicating that the drug may demonstrate substantial improvement over available therapies on one or more clinically significant endpoints. In the case of enfortumab vedotin, the designation was based on the initial results from Phase Ib/II EV-103 Clinical Trial.

The FDAs Breakthrough Therapy designation reflects the encouraging preliminary evidence for the combination of enfortumab vedotin and pembrolizumab in previously untreated advanced urothelial cancer to benefit patients who are in need of effective treatment options, said Andrew Krivoshik, M.D., Ph.D., Senior Vice President, and Oncology Therapeutic Area Head, Astellas.

We look forward to continuing our work with the FDA as we progress our clinical development program as quickly as possible.

This is an important step in our investigation of enfortumab vedotin in combination with pembrolizumab as first-line therapy for patients with advanced urothelial cancer who are unable to receive cisplatin-based chemotherapy, said Roger Dansey, M.D., Chief Medical Officer, Seattle Genetics.

Based on encouraging early clinical activity, we recently initiated a phase III trial of this platinum-free combination and look forward to potentially addressing an unmet need for patients.

Clinical trialThe Breakthrough Therapy designation was granted based on results from the dose-escalation cohort and expansion cohort A of the Phase Ib/II trial, EV-103 (NCT03288545), evaluating patients with locally advanced or metastatic urothelial cancer who are unable to receive cisplatin-based chemotherapy-treated in the first-line setting with enfortumab vedotin-ejfv in combination with pembrolizumab.

The initial results from the trial were presented at the European Society of Medical Oncology (ESMO) 2019 Congress, and updated findings at the 2020 Genitourinary Cancers Symposium.

EV-103 is an ongoing, multi-cohort, open-label, multicenter phase Ib/II trial of PADCEV alone or in combination, evaluating the safety, tolerability, and efficacy in muscle-invasive, locally advanced and first- and second-line metastatic urothelial cancer.

Adverse eventsSerious adverse reactions occurred in 46% of patients treated with enfortumab vedotin-ejfv. The most common serious adverse reactions (3%) were urinary tract infection (6%), cellulitis (5%), febrile neutropenia (4%), diarrhea (4%), sepsis (3%), acute kidney injury (3%), dyspnea (3%), and rash (3%). Fatal adverse reactions occurred in 3.2% of patients, including acute respiratory failure, aspiration pneumonia, cardiac disorder, and sepsis (each 0.8%).

Discontinuing treatmentAdverse reactions leading to discontinuation occurred in 16% of patients; the most common adverse reaction leading to discontinuation was peripheral neuropathy (6%). Adverse reactions leading to dose interruption occurred in 64% of patients; the most common adverse reactions leading to dose interruption were peripheral neuropathy (18%), rash (9%) and fatigue (6%). Adverse reactions leading to dose reduction occurred in 34% of patients; the most common adverse reactions leading to dose reduction were peripheral neuropathy (12%), rash (6%) and fatigue (4%).

The most common adverse reactions (20%) were fatigue (56%), peripheral neuropathy (56%), decreased appetite (52%), rash (52%), alopecia (50%), nausea (45%), dysgeusia (42%), diarrhea (42%), dry eye (40%), pruritus (26%) and dry skin (26%). The most common Grade 3 adverse reactions (5%) were rash (13%), diarrhea (6%) and fatigue (6%).

Specific recommendations

HyperglycemiaHyperglycemia occurred in patients treated with enfortumab vedotin-ejfv, including death and diabetic ketoacidosis (DKA), in patients with and without pre-existing diabetes mellitus. The incidence of Grade 3-4 hyperglycemia increased consistently in patients with higher body mass index and in patients with higher baseline A1C. In one clinical trial, 8% of patients developed Grade 3-4 hyperglycemia. Patients with baseline hemoglobin A1C 8% were excluded.

Physicians are recommended to closely monitor blood glucose levels in patients with, or at risk for, diabetes mellitus or hyperglycemia and, if blood glucose is elevated (>250 mg/dL), withhold the drug.

Peripheral neuropathyPeripheral neuropathy (PN), predominantly sensory, occurred in 49% of the 310 patients treated with enfortumab vedotin-ejf in clinical trials. Two percent (2%) of patients experienced Grade 3 reactions. In one clinical trial, peripheral neuropathy occurred in patients treated with enfortumab vedotin-ejf with or without preexisting peripheral neuropathy.

The median time to onset of Grade 2 was 3.8 months (range: 0.6 to 9.2). Neuropathy led to treatment discontinuation in 6% of patients. At the time of their last evaluation, 19% had complete resolution, and 26% had partial improvement.

Physicians should:

Occular disordersOcular disorders occurred in 46% of the 310 patients treated with enfortumab vedotin-ejf. The majority of these events involved the cornea and included keratitis, blurred vision, limbal stem cell deficiency and other events associated with dry eyes. Dry eye symptoms occurred in 36% of patients, and blurred vision occurred in 14% of patients, during treatment with enfortumab vedotin-ejf.

The median time to onset to symptomatic ocular disorder was 1.9 months (range: 0.3 to 6.2).

Physicians should monitor patients for ocular disorders and consider:

Skin reactionsSkin reactions occurred in 54% of the 310 patients treated with enfortumab vedotin-ejf in clinical trials. Twenty-six percent (26%) of patients had a maculopapular rash and 30% had pruritus. Grade 3-4 skin reactions occurred in 10% of patients and included symmetrical drug-related intertriginous and flexural exanthema (SDRIFE), bullous dermatitis, exfoliative dermatitis, and palmar-plantar erythrodysesthesia. In one clinical trial, the median time to onset of severe skin reactions was 0.8 months (range: 0.2 to 5.3).

Of the patients who experienced rash, 65% had complete resolution and 22% had partial improvement.

Physicians should monitor patients for skin reactions, and consider:

Infusion site extravasationSkin and soft tissue reactions secondary to extravasation have been observed after the administration of enfortumab vedotin-ejf. Of the 310 patients, 1.3% of patients experienced skin and soft tissue reactions. Reactions may be delayed.

Erythema, swelling, increased temperature, and pain worsened until 2-7 days after extravasation and resolved within 1-4 weeks of peak. One percent (1%) of patients developed extravasation reactions with secondary cellulitis, bullae, or exfoliation.

Physicians should ensure adequate venous access prior to starting enfortumab vedotin-ejf and monitor for possible extravasation during administration. If extravasation occurs, stop the infusion and monitor for adverse reactions.

Embryo-fetal toxicityEnfortumab vedotin-ejf can cause fetal harm when administered to a pregnant woman.

Physicians should advise patients of the potential risk to the fetus and advise female patients of reproductive potential to use effective contraception during enfortumab vedotin-ejf treatment and for 2 months after the last dose. At the same time, they should advise male patients with female partners of reproductive potential to use effective contraception during treatment with enfortumab vedotin-ejf and for 4 months after the last dose.

Clinical trialA Study of Enfortumab Vedotin Alone or With Other Therapies for Treatment of Urothelial Cancer (EV-103) NCT03288545

References[1] American Cancer Society. Cancer Facts & Figures 2020. Online. Last accessed on January 23, 2020.[2] American Society of Clinical Oncology. Bladder cancer: introduction (10-2017). Online. Last accessed on January 23, 2020.[3] International Agency for Research on Cancer. Cancer Tomorrow: Bladder. Online. Last accessed on January 23, 2020.[4] National Comprehensive Cancer Network (NCCN). NCCN Clinical Practice Guidelines in Oncology: Bladder Cancer. Version 4; July 10, 2019. Online. Last accessed on January 23, 2020.[5] Enfortumab vedotin-ejfv (Padcev; Astellas Pharma [package insert]. Northbrook, IL)[6] Challita-Eid P, Satpayev D, Yang P, et al. Enfortumab Vedotin Antibody-Drug Conjugate Targeting Nectin-4 Is a Highly Potent Therapeutic Agent in Multiple Preclinical Cancer Models. Cancer Res 2016;76(10):3003-13.

A version of this article was first published in ADC Review | Journal of Antibody-drug Conjugates.

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Combination Enfortumab Vedotin + Pembrolizumab Granted Breakthrough Therapy in Bladder Cancer - OncoZine

Vascular endotheliumtargeted Sirt7 gene therapy rejuvenates blood vessels and extends life span in a Hutchinson-Gilford progeria model – Science…

Abstract

Vascular dysfunction is a typical characteristic of aging, but its contributing roles to systemic aging and the therapeutic potential are lacking experimental evidence. Here, we generated a knock-in mouse model with the causative Hutchinson-Gilford progeria syndrome (HGPS) LmnaG609G mutation, called progerin. The Lmnaf/f;TC mice with progerin expression induced by Tie2-Cre exhibit defective microvasculature and neovascularization, accelerated aging, and shortened life span. Single-cell transcriptomic analysis of murine lung endothelial cells revealed a substantial up-regulation of inflammatory response. Molecularly, progerin interacts and destabilizes deacylase Sirt7; ectopic expression of Sirt7 alleviates the inflammatory response caused by progerin in endothelial cells. Vascular endotheliumtargeted Sirt7 gene therapy, driven by an ICAM2 promoter, improves neovascularization, ameliorates aging features, and extends life span in Lmnaf/f;TC mice. These data support endothelial dysfunction as a primary trigger of systemic aging and highlight gene therapy as a potential strategy for the clinical treatment of HGPS and age-related vascular dysfunction.

Aging represents the largest risk factor for many age-related diseases, as exemplified by cardiovascular diseases (CVDs) (1). The blood vessel consists of the tunica intima [composed of endothelial cells (ECs)], the tunica media [composed of vascular smooth muscle cells (VSMCs)], and the tunica adventitia (consisting of connective tissue) (2). The endothelium separates the vessel wall from blood flow and has an irreplaceable role in regulating vascular tone and homeostasis. Age-related functional decline in ECs and VSMCs is a main cause of CVDs (3). ECs secrete various vasodilators and vasoconstrictors that act on VSMCs and induce blood vessel contraction and relaxation (4). For instance, nitric oxide (NO) is synthesized from l-arginine by endothelial NO synthase (eNOS) and then released on VSMCs to induce blood vessel relaxation (5). When ECs become senescent or dysfunctional, vasoconstrictive, procoagulative, and proinflammatory cytokines are released; this effect reduces NO bioavailability and, in turn, increases vascular intimal permeability and EC migration (6). Despite advances in the understanding of mechanisms of endothelial dysfunction, it is unclear whether it directly triggers organismal aging.

Accumulating evidences suggest that the mechanisms underlying physiological aging are similar to those governing Hutchinson-Gilford progeria syndrome (HGPS)a premature aging syndrome in which affected patients typically succumb to CVDs (7). HGPS is predominantly caused by an a.c. 1824 C>T, p. G608G mutation in LMNA gene, which activates an alternate splicing event and generates a 50amino acid truncated form of Lamin A, referred to as progerin (8). The murine LmnaG609G, which is equivalent to LMNAG608G in humans, causes aging phenotypes resembling HGPS (9). It has been shown that progerin targets SMCs and causes blood vessel calcification and atherosclerosis (10, 11). Recent work by two groups showed that SMC-specific progerin knock-in (KI) mice are healthy and have a normal life span but suffer from blood vessel calcification, atherosclerosis, and shortened life span when crossed to Apoe/ mice (12, 13). In contrast to SMCs, the contributing roles of the vascular endothelium (VE) to systemic/organismal aging are still elusive. To address these issues, we generated a conditional progerin (LmnaG609G) KI model, i.e., Lmnaf/f mice. In combination with E2A-Cre and Tie2-Cre mice, in which the expression of Cre is ubiquitous including germ cells (14) or driven by the endothelial-specific Tie2 promoter (15), we aimed to investigate the roles of VE dysfunction to systemic aging and the targeting potential for the clinical treatment of HGPS.

To study the mechanism of VE aging, we generated a mouse model of conditional progerin KI, in which the LmnaG609G mutation, equivalent to HGPS LMNAG608G, was flanked with loxP sites, i.e., Lmnaf/f mice (fig. S1A). The Lmnaf/f mice were crossed to E2A-Cre mice, in which the Cre recombinase is ubiquitously expressed including germ cells, to generate LmnaG609G/G609G and LmnaG609G/+ mice. Progerin was ubiquitously expressed in LmnaG609G/G609G and LmnaG609G/+ mice, which recapitulated many progeroid features found in HGPS, including growth retardation and shortened life span (fig. S1, B to D).

To understand primary alterations in the VE, we isolated CD31+ murine lung ECs (MLECs) (16) from three pairs of LmnaG609G/G609G (G609G) and Lmnaf/f (Flox) mice by fluorescence-activated cell sorting (FACS) (Fig. 1A) and performed 10 Genomics single-cell RNA sequencing. We recovered 6004 cells (4137 from G609G and 1867 from Flox mice) and used the k-means clustering algorithm to cluster the cells into four groups (Fig. 1B). As expected, one group exhibited high Cd31, Cd34, and Cdh5 expression and thus largely represented MLECs. The other three groups, copurified with CD31+ MLECs by FACS, showed relatively lower Cd31 expression at the mRNA level (>10-fold lower than MLECs) but high Cd45 expression (fig. S2). Further analysis revealed that these clusters most likely contained B lymphocytes (B-like) with high Cd22, Cd81, and Ly6d expression; T lymphocytes (T-like) with high Cd3d, Cd3e, and Cd28 expression; and macrophages (M-like) with high Cd14, Cd68, and Cd282 expression (Fig. 1C). Most of the marker gene expression levels were comparable between G609G and Flox mice, except for Cd34 and Icam1, which were significantly elevated in G609G ECs, and Cd14 and Vcam1, which were increased in G609G M-like cells (Fig. 1D). Of note, Icam1 and Vcam1 are among the most conserved markers of endothelial senescence and atherosclerosis (17). Thus, we established an Lmnaf/f conditional progerin KI mouse model and revealed a unique EC population for mechanistic study.

(A) Purity analysis of sorted CD31+ MLECs by FACS. SSC, side scatter; FSC, forward scatter; PE, phycoerythrin. (B) t-Distributed stochastic neighbor embedding (t-SNE) projection of CD31+ cells revealed four clusters: ECs (green), B lymphocytes (B-like; orange), T lymphocytes (T-like; blue), and macrophages (M-like; red). (C) Marker gene expression in the four clusters: ECs (Cd31, Cd34, and Cdh5), B-like (Ly6d, Cd22, and Cd81), T-like (Cd3d, Cd3e, and Cd28), and M-like (Cd14, Cd68, and Cd282). (D) Heatmap showing marker gene expression levels in LmnaG609G/G609G (G609G) and Lmnaf/f (Flox) mice.

Of the four clusters of CD31+ MLECs, ECs and M-like cells showed high levels of p21Cip1/Waf1 (fig. S2A), a typical senescence marker (18). This finding suggests that these cells are the main target of progerin in the context of aging. A previous study reported that M-specific progerin, achieved by crossing Lmnaf/+ to Lyz-Cre mice, caused minimal aging phenotypes (12), implicating that M might have only a minor role in organismal aging. We thus focused on ECs for further analysis. We recovered 899 and 445 ECs from E2A and Flox mice, respectively (Fig. 2A). Genes with >1.5-fold change in expression between these mice were chosen for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. We observed a significant enrichment in the pathways that regulate chemotaxis, immune responses in malaria and Chagas diseases, inflammatory bowel disease, and rheumatoid arthritis and pathways essential for cardiac function (Fig. 2, B to D). To confirm this observation and to exclude paracrine effects from other cell types, we overexpressed progerin in human umbilical vein ECs (HUVECs) and analyzed representative genes by quantitative polymerase chain reaction (PCR). Most of the examined genes, e.g., IL6, IL8, IL15, CXCL1, IL1, etc., were significantly up-regulated upon ectopic progerin overexpression (Fig. 2E). Together, these data suggest that progerin causes an inflammatory response in VE, which might lead to systemic aging.

(A) t-SNE projection of LmnaG609G/G609G (G609G; green) and Lmnaf/f (Flox; orange) CD31+ MLECs according to transcriptomic data. (B to D) GO and KEGG pathway enrichment of differentially expressed genes between G609G and Flox cells. LmnaG609G/G609G MLECs show enrichment in genes that regulate the inflammatory response (C) and genes related to heart dysfunction (D). FC, fold change; FDR, false discovery rate. (E) Quantitative PCR analysis of altered genes observed in (C) and (D) in HUVECs with ectopic expression of progerin or wild-type LMNA. Data represent means SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 (Students t test).

To test whether the VE dysfunction has essential roles in systemic aging, we crossed Lmnaf/f mice to a Tie2-Cre line to generate Lmnaf/f;TC mice, in which the expression of Cre recombinase is driven by the promoter/enhancer of endothelial-specific Tie2 gene (15). Single-cell transcriptome analysis confirmed that Tie2 was mainly detected in ECs (fig. S2B). Consistently, progerin was observed in the VE of Lmnaf/f;TC, but not in that of Lmnaf/f control mice or other tissues (fig. S3). VE-specific progerin induced intima-media thickening in Lmnaf/f;TC mice, in a similar manner to total KI mice, i.e., LmnaG609G/G609G mice (Fig. 3, A and B). We performed functional analysis of the VE based on acetylcholine (ACh)regulated vasodilation. ACh-induced thoracic aorta relaxation was significantly compromised in Lmnaf/f;TC mice (Fig. 3C). Similar defects were observed in LmnaG609G/G609G and LmnaG609G/+ mice (Fig. 3D and fig. S4), where progerin was expressed in both ECs and SMCs (12). To gain more evidence supporting VE-specific dysfunction, we examined thoracic aorta relaxation induced by sodium nitroprusside (SNP), which is an SMC-dependent vasodilator. Little difference was observed in thoracic aorta vasodilation in LmnaG609G/G609G and LmnaG609G/+ compared to Lmnaf/f control mice (Fig. 3E and fig. S4), supporting the notion that the VE dysfunction is a key contributor of vasodilation defects in progeria mice. As NO is the most potent vasodilator (19), we examined eNOS levels in the thoracic aorta of Lmnaf/f;TC and Lmnaf/f control mice. As expected, the level of eNOS was significantly reduced in Lmnaf/f;TC mice compared to Lmnaf/f control mice (Fig. 3F). Thus, the data confer a VE-specific dysfunction in progeria mice.

(A and B) Hematoxylin and eosin staining of thoracic aorta sections from (A) Lmnaf/f;TC and (B) LmnaG609G/G609G and Lmnaf/f control mice showing intima-media thickening. Scale bar, 20 m. (C) ACh-induced thoracic aorta vasodilation in Lmnaf/f;TC and Lmnaf/f control mice. **P < 0.01. 5-HT, 5-hydroxytryptamine. (D) ACh-induced thoracic aorta vasodilation in LmnaG609G/G609G and control mice. **P < 0.01. (E) SNP-induced thoracic aorta vasodilation in LmnaG609G/G609G and control mice. (F) eNOS level in thoracic aorta sections from Lmnaf/f;TC and control mice. Scale bar, 20 m. (G) Immunofluorescence staining (left) and quantification (right) of CD31+ gastrocnemius muscle in Lmnaf/f;TC and Lmnaf/f mice. Scale bar, 50 m. DAPI, 4,6-diamidino-2-phenylindole. (H) CD31 immunofluorescence staining in Lmnaf/f;TC and Lmnaf/f liver. Scale bar, 50 m. (I) Representative microcirculation images (left) and quantification of blood flow recovery (right) following hindlimb ischemia in Lmnaf/f;TC and Lmnaf/f mice. (J) Representative transverse sections and quantification of CD31+ gastrocnemius muscle 14 days after femoral artery ligation. Scale bar, 50 m. All data represent means SEM. P values were calculated by Students t test. Photo credits: Shimin Sun, School of Life Sciences, Shandong University of Technology; Medical Research Center, Shenzhen University (A, B, F, H, and J); Weifeng Qin, Medical Research Center, Shenzhen University (G and I).

The reduced capillary density and neovascularization capacity are both characteristics of endothelial dysfunction (1). We examined the microvasculature in various tissues of Lmnaf/f;TC mice by immunofluorescence staining. We observed a significant loss in CD31+ ECs in Lmnaf/f;TC mice compared to controls (Fig. 3, G and H). We further examined ischemia-induced neovascularization ability in Lmnaf/f;TC mice following femoral artery ligation. Limb perfusion after ischemia was significantly blunted in Lmnaf/f;TC mice compared to controls (Fig. 3I). Histological analysis confirmed that the defect in blood flow recovery in Lmnaf/f;TC mice was a reflection of an impaired ability to form new blood vessels in the ischemic region (Fig. 3J). Together, Lmnaf/f;TC mice are characterized by a loss of ECs, a reduced capillary density, and defective neovascularization capacity.

The single-cell transcriptome implicates heart dysfunction in LmnaG609G/G609G mice (Fig. 2). A correlation with gene alterations associated with atherosclerosis and osteoporosis was obvious in LmnaG609G/G609G ECs (the Online Mendelian Inheritance in Man; https://omim.org) (fig. S5). We thus reasoned that endothelial-specific dysfunction might be enough to trigger systemic aging. Notably, atherosclerosis was prominent in Lmnaf/f;TC mice (aorta atheromatous plaque observed in all nine examined mice; Fig. 4A), as well as severe fibrosis in the arteries and hearts (Fig. 4, B and C); both are typical features of aging. Moreover, the heart/body weight ratio was significantly increased in Lmnaf/f;TC compared to Lmnaf/f control mice (Fig. 4D), indicating dilated cardiomyopathy (20). Echocardiography confirmed that heart rate, cardiac output, left ventricular ejection fraction, and fractional shortening were significantly reduced in 7- to 8-month-old Lmnaf/f;TC compared to Lmnaf/f control mice. The running endurance was largely compromised in Lmnaf/f;TC mice (Fig. 4E), which is likely a reflection of amyotrophy. Moreover, the microcomputed tomography (CT) identified a decrease in trabecular bone volume/tissue volume, trabecular thickness, and trabecular number but an increase in trabecular separation in Lmnaf/f;TC mice (Fig. 4F), indicative of osteoporosis, which is an important hallmark of systemic aging (21). The VE-specific dysfunction not only accelerated aging in various tissues/organs but also shortened the median life span of Lmnaf/f;TC mice (24 weeks) to a similar extent to LmnaG609G/G609G mice (21 weeks) (Fig. 4G). LmnaG609G/G609G mice suffered from body weight loss roughly from 8 weeks of age, while Lmnaf/f;TC mice only showed a slight drop in body weight (Fig. 4H), suggesting that body weight loss itself is a less likely primary causal factor to progeria compared to endothelial dysfunction. Together, these results implicate that endothelial dysfunction, at least in progeria, acts as a causal factor of systemic aging.

(A to C) Masson trichrome staining showing an atheromatous plaque in the aorta (A), SMC loss (B), and cardiac fibrosis (C) in Lmnaf/f;TC mice. Scale bar, 20 m. (D) Heart weight and echocardiographic parameters, including heart rate, cardiac output, left ventricular (LV) ejection fraction (LVEF), and left ventricular ejection shortening (LVFS). *P < 0.05, Lmnaf/f;TC versus Lmnaf/f mice. (E) Decreased running endurance in Lmnaf/f;TC mice. ***P < 0.001. (F) Micro-CT analysis showing a decrease in trabecular bone volume/tissue volume (BV/TV), trabecular number, and trabecular thickness and an increase in trabecular separation in Lmnaf/f;TC mice. *P < 0.05, Lmnaf/f;TC versus Lmnaf/f mice. (G) Life span of LmnaG609G/G609G, LmnaG609G/+, Lmnaf/f;TC, and Lmnaf/f mice. (H) Body weight of male LmnaG609G/G609G, LmnaG609G/+, Lmnaf/f;TC, and Lmnaf/f mice. *P < 0.05, Lmnaf/f;TC versus Lmnaf/f mice; ***P < 0.001, LmnaG609G/G609G versus Lmnaf/f mice. All data represent means SEM. P values were calculated by Students t test, except that statistical comparison of the survival data was performed by log-rank test. Photo credits: Weifeng Qin, Medical Research Center, Shenzhen University (A and B); Shimin Sun, School of Life Sciences, Shandong University of Technology; Medical Research Center, Shenzhen University (C).

Loss of Sirt7, an NAD+ (nicotinamide adenine dinucleotide)dependent deacylase, causes heart dysfunction with systemic inflammation and accelerates aging (22, 23). We noticed defective neovascularization in Sirt7 knockout mice (Fig. 5A). Knockdown of Sirt7 up-regulated the levels of interleukin-1 (IL-1) and IL6 in HUVECs, as determined by Western blotting and real-time PCR (Fig. 5, B and C). Significantly, the protein level of Sirt7 was reduced almost 50% in Lmnaf/f;TC MLECs (Fig. 5D). By contrast, the levels of Sirt6 and Sirt1 were hardly decreased in Lmnaf/f;TC MLECs. Furthermore, co-immunoprecipitation revealed that Lamin A interacted with Sirt7, which was significantly enhanced in the case of progerin (Fig. 5E). FLAG-SIRT7 was polyubiquitinated, which was enhanced in the presence of progerin compared with Lamin A (Fig. 5F). Ectopic expression of progerin in human embryonic kidney (HEK) 293 accelerated SIRT7 protein degradation, which was inhibited by MG132 (a proteasome inhibitor) (Fig. 5G). These data suggest that accumulation of progerin destabilizes Sirt7 by proteasomal pathway in progeria cells.

(A) Quantification of blood flow recovery following hindlimb ischemia in Sirt7/ and Sirt7+/+ mice. (B) Left: Representative immunoblots showing indicated protein levels in HUVECs treated with si-SIRT7 or scramble (Scram). Right: Quantification of relative protein levels. *P < 0.05 and **P < 0.01, small interfering RNA (siRNA) versus Scram. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C) Real-time PCR analysis of the indicated gene expression in HUVECs treated with si-SIRT7 or Scram. *P < 0.05, siRNA versus Scram. (D) Left: Representative immunoblots showing indicated sirtuin protein levels in FACS-sorted MLECs. Right: Quantification of relative protein levels. *P < 0.05. Note that down-regulated Sirt7 but rather up-regulated Sirt6 and hardly changed SIRT1 in Lmnaf/f;TC MLECs. (E) Left: Co-immunoprecipitation (IP) experiments showing hemagglutinin (HA)SIRT7 in antiFLAGLamin A and antiFLAG-progerin immunoprecipitates. Right: Quantification of relative protein levels. *P < 0.05. (F) Left: Representative immunoblots showing polyubiquitinated SIRT7, which was up-regulated in the presence of progerin but rather down-regulated in the presence of Lamin A. Right: Quantification of relative protein levels. *P < 0.05. (G) Representative immunoblots showing SIRT7 protein levels in the presence of Lamin A or progerin in HEK293 cells treated with cycloheximide (CHX) and/or MG132 (M). Quantification of relative SIRT7 protein levels was shown. *P < 0.05, progerin versus Lamin A. All data represent means SEM. P values were calculated by Students t test. Photo credit: Xiaolong Tang, Medical Research Center, Shenzhen University (B, D, E, F, and G).

We reasoned that Sirt7 might underlie the VE dysfunction in progeria mice. To test this hypothesis, we first examined whether ectopic Sirt7 could rescue the exacerbated inflammatory response in HUVECs. As shown, overexpression of SIRT7 significantly down-regulated the expression of multiple inflammatory genes such as IL1 (Fig. 6A). To test the in vivo function of Sirt7 in defective neovascularization, we generated a recombinant AAV serotype 1 (rAAV1) cassette with Sirt7 gene expression driven by a synthetic ICAM2 promoter (IS7O), which ensures VE-specific expression (24, 25). As shown, on-site injection of IS7O at a dose of 1.25 1010 viral genome-containing particles (vg)/50 l significantly improved blood vessel formation in Lmnaf/f;TC mice (Fig. 6B). The ectopic expression of Sirt7 and the increase in CD31-labeled ECs were evidenced by fluorescence confocal microscopy in ECs of regenerated blood vessels (Fig. 6, C and D).

(A) Real-time PCR analysis of genes that are aberrantly up-regulated in progerin-overexpressing HUVECs upon overexpression of SIRT7. *P < 0.05, **P < 0.01, and ***P < 0.001. (B) Neovascularization assay in Lmnaf/f;TC mice with hindlimb ischemia, treated with or without IS7O particles. **P < 0.01. (C) Immunofluorescence microscopy analysis of FLAG-SIRT7 and CD31 expression in gastrocnemius muscle 14 days after femoral artery ligation. Scale bar, 25 m. (D) Percent CD31+ ECs in Lmnaf/f;TC mice treated with or without IS7O particles. ***P < 0.001. (E) Representative immunofluorescence images of the liver, aorta, and muscle of Lmnaf/f;TC mice after IS7O therapy, showing CD31+ ECs with FLAG-SIRT7 expression. Scale bar, 50 m. (F) Representative immunoblots showing expression of FLAG-SIRT7 in aorta and WBMCs. Note that FLAG-SIRT7 was merely detected in WBMCs. (G) Life span of IS7O-treated and untreated Lmnaf/f;TC and LmnaG609G/+ mice. (H) Body weight of IS7O-treated and untreated Lmnaf/f;TC and Lmnaf/f mice. All data represent means SEM. P values were calculated by Students t test, except that the statistical comparison of survival data was performed by log-rank test. Photo credits: Shimin Sun, School of Life Sciences, Shandong University of Technology; Medical Research Center, Shenzhen University (C and E); Xiaolong Tang, Medical Research Center, Shenzhen University (F).

We next asked whether IS7O could ameliorate premature aging and extend life span. To this end, the IS7O particles were injected via tail vein from 21 weeks of age, when progeria mice start to die. The injection was repeated every other week at a concentration of 5 1010 vg/200 l per mouse. While all untreated mice died before 34 weeks of age, most IS7O-treated mice were still alive at the age of 44 weeks, when they were euthanized for histological analysis. The ectopic expression of FLAG-SIRT7 was observed in the ECs of liver, muscle, and aorta, but not in whole bone marrow cells (WBMCs), determined by fluorescence microscopy and/or Western blotting (Fig. 6, E and F). The median life span was extended by 76%from 25 to >44 weeks (Fig. 6G). The age-related body weight loss was slightly rescued upon IS7O therapy in Lmnaf/f;TC mice (Fig. 6H). These data suggest that progerin-caused VE dysfunction and systemic aging are partially, if not entirely, attributable to Sirt7 decline.

Mounting evidence supports the idea that endothelial dysfunction is a conspicuous marker for vascular aging and CVDs (2628). However, the fundamental question whether VE dysfunction causally triggers systemic aging remains. The heterogeneity of vascular cells and their close communication with the bloodstream render it difficult to understand the primary function of the VE. The murine LmnaG609G mutation, equivalent to the LMNAG608G found in humans with HGPS, causes premature aging phenotypes in various tissues and organs, thus providing an ideal model for studying aging mechanisms at both tissue and organismal levels. Data from the LmnaG609G model suggest that SMCs are the primary cause of vascular diseases, such as atherosclerosis (10, 11). A recent study showed that specific expression of LmnaG609G in SMCs causes atherosclerosis and shortens life span in atherosclerosis-prone Apoe/ mice (12). We used Tie2-Cre line to generate the VE-specific LmnaG609G mouse model. Lmnaf/f;TC mice exhibited vascular dysfunction, accelerated aging, and a shortened life span to a similar extent to the whole-body LmnaG609G model. Tie2 expression was reported not only in ECs but also in hematopoietic lineages (29). Our single-cell transcriptomic data identified Tie2 transcripts mainly in MLECs instead of B-, T-, or M-like cells. When a synthetic ICAM2 promoter was used to drive ectopic expression of FLAG-SIRT7 in the rescue experiments, ectopic FLAG-SIRT7 was successfully detected in ECs of the aorta, muscle, and liver but hardly detected in WBMCs. Therefore, Tie2-driven progerin expression combined with synthetic ICAM2-drivern SIRT7 rescue largely ensures the EC-specific contribution in systemic aging. Of note, although the number and function of hematopoietic stem cells decline in another progeria model, Zmpste24/ mice (30), little effect was observed when healthy hematopoietic progenitor cells were transplanted to Zmpste24/ mice in the context of systemic aging. Recently, Hamczyk et al. (12) found that knocking in the LmnaG609G allele in macrophages mediated by LysM-Cre merely affects aging and life span. Therefore, our data strongly suggest that, as the largest secretory organ (3), VE is pivotal in regulating systemic aging and longevity. In support of our findings, Foisner et al. (31) reported that VE-cadherin promoter-driven expression of progerin in a transgenic line causes cardiovascular abnormalities and shortens life span.

One limitation in the understanding of mechanisms of VE dysfunction is the vascular cell heterogeneity and the lack of appropriate in vitro system for ECs. Here, we took advantage of single-cell RNA sequencing technique to analyze the transcriptomes of MLECs. Unexpectedly, although >95% purity was achieved by FACS, MLECs isolated by CD31 immunofluorescence labeling turned out to be a mixture of cells, including ECs and T-, B-, and M-like cells. Although enriched by FACS, these non-ECs expressed low level of CD31 mRNA, raising the possibility that cell surface proteins such as CD31 T-, B-, and M-like cells might be obtained from neighbor ECs via intercellular protein transfer (32). Nevertheless, these findings suggest that one cannot just purify CD31+ cells and pool them together for mechanistic study, because one might arrive at a misleading conclusion. We compared the expression of genes that are associated with atherosclerosis, arthritis, heart failure, osteoporosis, or amyotrophy (the Online Mendelian Inheritance in Man; https://omim.org) between progeroid and control in all four clusters. An obvious alteration of these genes/pathways was observed mainly in ECs and M-like cells (fig. S2). At the current stage, it is hard to separate cell-autonomous and paracrine effects among different cell populations. In the future, it would be worthwhile to do an analysis in Lmnaf/f;TC MLECs. The data will be useful to study the paracrine effect of ECs on other cell populations.

Since the identification of the causal link between LMNA G608G mutation and HGPS, numerous efforts have been put on the development of treatment for HGPS. Farnesyltransferase inhibitors (33), resveratrol, and N-acetyl cysteine (30) treatment alleviate premature aging features and extend life span in progeria murine models. Rapamycin (34) and metformin (35) incubation rescue senescence in HGPS cells. On the basis of these notions, patients with HGPS taking a farnesyltransferase inhibitor, lonafarnib, in a clinical trial showed significant improvement of health status, reduction of mortality rate, and a potential extension of life span (about 1 to 2 years) (36). Taking advantage of gene therapy and the dispensable role of Lamin A, morpholino oligos (9), and CRISPR-Cas9 designs (37, 38), which prevent Lamin A/progerin generation, can alleviate aging features and extend life span from 25 to 40% in progeria mice. However, considering the indispensable function of Lamin A in humans, these genome-modifying strategies need further experimentation before potential clinical application. Here, applying a different strategy, we showed that rAAV1-SIRT7 (IS7O), targeting dysfunctional VE, largely ameliorates progeroid features and almost doubles the median life span (from 25 to >44 weeks). To our best knowledge, this is the most marked rescue of progeria in a mouse model via gene therapy. Given that SIRT7 elicits deacylase activity to modulate cellular functions (22, 23), it is worthwhile to identify small molecules that specifically target SIRT7 activity for therapeutics in the future. Resveratrol is a potential activator of SIRT1, as well as SIRT7 (39), and has protective effects on vascular function and blood pressure (40). Further depicting the relationship of SIRT7 and resveratrol in the regulation of vascular function would help in seeking leading compounds of SIRT7 specific activators.

Collectively, we reveal VE dysfunction as a primary trigger of systemic aging and as a risk factor for age-related diseases such as atherosclerosis, heart failure, and osteoporosis. Drugs and molecules that target VE might serve as good candidates in the treatment of age-related diseases other than CVDs. The findings in SIRT7-based gene therapy implicate great clinical potentials for progeria as well as in antiaging applications.

Lmnaf/+ allele (LmnaG609G mutation flanked by two loxP sites) was generated by Cyagen Biosciences Inc., China. Briefly, the 5 and 3 homology arms were amplified from bacterial artificial chromosome clones RP23-21K15 and RP23-174J9, respectively. The G609G (GGC to GGT) mutation was introduced into exon 11 in the 3 homology arm. C57BL/6 embryonic stem cells were used for gene targeting. To obtain ubiquitous expression of progerin (LmnaG609G/G609G), Lmnaf/f mice were bred with E2A-Cre mice. To obtain VE-specific expression of progerin, Lmnaf/f mice were bred with Tie2-cre mice. Mice were housed and handled in accordance with protocols approved by the Committee on the Use of Live Animals in Teaching and Research of Shenzhen University, China.

Four-month-old male mice were anesthetized with 4% chloral hydrate (0.20 ml/20 g) by intraperitoneal injection. Hindlimb ischemia was performed by unilateral femoral artery ligation and excision, as previously described (41). In brief, the neurovascular pedicle was visualized under a light microscope following a 1-cm incision in the skin of the left hindlimb. Ligations were made in the left femoral artery proximal to the superficial epigastric artery branch and anterior to the saphenous artery. Then, the femoral artery and the attached branches between ligations were excised. The skin was closed using a 4-0 suture line, and erythromycin ointment was applied to prevent wound infection after surgery. Recovery of the blood flow was evaluated before and after surgery using a dynamic microcirculation imaging system (Teksqray, Shenzhen, China). Relative blood flow recovery is expressed as the ischemia-to-nonischemia ratio. At least three mice were included in each experimental group.

HEK293 cells and HUVECs were purchased from the American Type Culture Collection. HEK293 cells were cultured in Gibco Dulbeccos modified Eagles medium (Life Technologies, USA) supplemented with 10% fetal bovine serum at 37C, 5% CO2. HUVECs were cultured in Gibco M199 (Life Technologies, USA) supplemented with 15% fetal bovine serum, EC growth supplement (50 g/ml), and heparin (100 g/ml) at 37C, 5% CO2. All cell lines used were authenticated by short tandem repeat profile analysis and were mycoplasma free.

Total RNA was extracted from cells or mouse tissues using TRIzol reagent RNAiso Plus (Takara, Japan) and transcribed into complementary DNA (cDNA) using 5 PrimeScript RT Master Mix (Takara, Japan), following the manufacturers instructions. The mRNA levels were determined by quantitative PCR with SYBR Premix Ex Taq II (Takara, Japan) detected on a CFX Connect Real-Time PCR Detection System (Bio-Rad). All primer sequences are listed in table S1.

For protein extraction, cells were suspended in SDS lysis buffer and boiled. Then, the lysate was centrifuged at 12,000g for 2 min, and the supernatant was collected. For Western blotting, protein samples were separated on SDS-polyacrylamide gels, transferred to polyvinylidene difluoride membranes (Millipore, USA), blocked with 5% nonfat milk, and incubated with the relevant antibodies. Images were acquired on a Bio-Rad system. All antibodies are listed in table S2.

Frozen sections of aorta, skeletal muscle, and liver tissues were fixed in 4% paraformaldehyde (PFA), permeabilized with 0.3% Triton X-100, blocked with 5% bovine serum albumin and 1% goat serum, and then incubated with primary antibodies at room temperature for 2 hours or at 4C overnight. After three washes with phosphate-buffered saline with Tween 20, the sections were incubated with secondary antibodies for 1 hour at room temperature and then stained with 4,6-diamidino-2-phenylindole antifade mounting medium. Images were captured under a Zeiss LSM 880 confocal microscope. All antibodies are listed in table S2.

Paraffin-embedded sections of PFA-fixed tissues were dewaxed and hydrated. Staining was then performed using a Masson trichrome staining kit (Beyotime, China). In brief, the sections were dipped in Bouin buffer for 2 hours at 37C and then successively stained with Celestine blue staining solution, hematoxylin staining solution, Ponceau S staining solution, and aniline blue solution for 3 min. After dehydrating with ethyl alcohol three times, the sections were mounted with Neutral Balsam Mounting Medium (BBI Life Science, China). Images were captured under a Zeiss LSM 880 confocal microscope.

Mice were euthanized by decapitation. The lungs were then collected, cut into small pieces, and then digested with collagenase I (200 U/ml) and neutral protease (0.565 mg/ml) for 1 hour at 37C. The isolated cells were incubated with phycoerythrin-conjugated anti-CD31 antibody for 1 hour at 4C and then 7-aminoactinomycin D (7-AAD) (1:100) for 5 min. CD31-positive and 7-AADnegative cells were sorted on a flow cytometer (BD Biosciences, USA).

Four-month-old male mice were anesthetized with 4% chloral hydrate by intraperitoneal injection. Thoracic aortas were collected, rinsed in ice-cold Krebs solution, and cut into 2-mm-length rings. Each aorta ring was bathed in 5-ml oxygenated (95% O2 and 5% CO2) Krebs solution at 37C for 30 min in a myograph chamber (620M, Danish Myo Technology). Each ring was stretched in a stepwise fashion to the optimal resting tension (thoracic aortas to ~9 mN) and equilibrated for 30 min. Then, 100 mM K+ Krebs solution was added to the chambers to elicit a reference contraction and then washed out with Krebs solution at 37C until a baseline was achieved. Vasodilation induced by Ach or SNP (1 nM to 100 M) was recorded in 5-hydroxytryptamine (2 M) contracted rings. Data are represented as a percentage of force reduction and the peak of K+-induced contraction. At least three mice were included in each experimental group.

Seven- to 8-month-old male mice were anesthetized by isoflurane gas inhalation and then subjected to transthoracic echocardiography (iU22, Philips). Parameters, including heart rate, cardiac output, left ventricular posterior wall dimension, left ventricular end-diastolic dimension, left ventricular end-systolic diameter, LV ejection fraction, and LV fractional shortening, were acquired. At least three mice were included in each experimental group.

Seven- to 8-month-old male mice were euthanized by decapitation. The thigh bone was fixed in 4% PFA at 4C overnight. The relevant data were collected by micro-CT (Scanco Medical, CT100). At least three mice were included in each experimental group.

A Rota-Rod Treadmill (YLS-4C, Jinan Yiyan Scientific Research Company, China) was used to monitor fatigue resistance. Briefly, mice were placed on the rotating lane, and the speed of the rotations gradually increased to 40 rpm. When the mice were exhausted, they were safely dropped from the rotating lane, and the latency to fall was recorded. At least three mice were included in each experimental group.

CD31+ cells isolated from murine lung by FACS (>90% viability) were used for single-cell RNA sequencing. A sequence library was built according to the Chromium Single-Cell Instrument library protocol (42). Briefly, single-cell RNAs were barcoded and reverse-transcribed using the Chromium Single-Cell 3 Reagent Kits v2 (10 Genomics) and then fragmented and amplified to generate cDNAs. The cDNAs were quantified using an Agilent Bioanalyzer 2100 DNA Chip, and the library was sequenced using an Illumina Hiseq PE150 with ~10 to 30M raw data assigned for each cell. The reads were mapped to the mouse mm9 genome and analyzed using STAR: >90% reads mapped confidently to genomic regions and >50% mapped to exonic regions. Cell Ranger 2.1.0 was used to align reads, generate feature-barcode matrices, and perform clustering and gene expression analysis. Mean reads (>80,000) and 900 median genes per cell were obtained. The unique molecular identifier counts were used to quantify the gene expression levels, and the t-distributed stochastic neighbor embedding (t-SNE) algorithm was used for dimensionality reduction. The cell population was then clustered by k-means clustering (k = 4). The Log2FoldChange was the ratio of gene expression of one cluster to that of all other cells. The P value was calculated using the negative binomial test, and the false discovery rate was determined by the Benjamini-Hochberg procedure. GO and KEGG enrichment analyses were performed in DAVID version 6.8 (43).

A two-tailed Students t test was used to determine statistical significance, except that the statistical comparison of survival data was performed by log-rank test. All data are presented as the means SD or means SEM, as indicated, and a P value <0.05 was considered statistically significant.

Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/8/eaay5556/DC1

Fig. S1. Generation of Lmnaf/f mice and phenotypic analysis of LmnaG609G/G609G mice.

Fig. S2. Single-cell transcriptomic analysis of CD31+ MLECs.

Fig. S3. VE-specific progerin expression.

Fig. S4. Vasodilation analysis of LmnaG609G/+ mice.

Fig. S5. Expression of atherosclerosis- and osteoporosis-associated genes in MLEC transcriptomes.

Table S1. List of primer sequences.

Table S2. List of antibodies.

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

Acknowledgments: We thank J. Tamanini (Shenzhen University and ETediting) for editing the manuscript before submission. Funding: This study was supported by grants from the National Natural Science Foundation of China (91849208, 81571374, 91439133, 81871114, 81601215, 81972602, and 81702909), the National Key R&D Program of China (2017YFA0503900), the Science and Technology Program of Guangdong Province (2014A030308011, 2017B030301016, and 2019B030301009), and the Shenzhen Municipal Commission of Science and Technology Innovation (JCYJ20160226191451487, KQJSCX20180328093403969, JCYJ20180507182044945, ZDSYS20190902093401689, and Discipline Construction Funding of Shenzhen 2016-1452). Author contributions: B.L. designed and supervised the project. S.S., W.Q., and X.T. conducted experiments with help from W.H., S.Z., M.Q., Z.L., X.C., Q.P., and B.Z. Y.M. performed bioinformatic analysis. Z.W. and Z.Z. provided resources. S.S., X.T., and B.L. wrote the manuscript. All authors discussed the experimental results and reviewed the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. The data of single-cell transcriptomics are available in the GEO database (GSE138975). Additional data related to this paper may be requested from the authors.

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