The effects of losartan or angiotensin II receptor antagonists on cartilage: a systematic review.

OBJECTIVE: The aim of this study is to analyze the latest evidence on the effects of losartan or Ang II receptor antagonists on cartilage repair, with a focus on their clinical relevance. DESIGN: The PubMed, Embase, and Cochrane Library databases were searched up to November 12th 2021 to evaluate the effects of losartan or Ang II receptor antagonists on cartilage repair in in vitro studies and in vivo animal studies. Study design, sample characteristics, treatment type, duration, and outcomes were analyzed. The risk of bias and the quality of the eligible studies were assessed using the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) risk of bias assessment tool and Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Studies (CAMARADES). RESULTS: A total of 12 studies were included in this systematic review. Of the 12 eligible studies, two studies were in vitro human studies, three studies were in vitro animal studies, one study was an in vitro human and animal study, and six studies were in vivo animal studies. The risk bias and quality assessments were predominantly classified as moderate. Since meta-analysis was difficult due to differences in treatment type, dosage, route of administration, and method of outcome assessment among the eligible studies, qualitative evaluation was conducted for each study. CONCLUSIONS: Both in vitro and in vivo studies provide evidence to demonstrate beneficial effects of Ang II receptor antagonists on osteoarthritis and cartilage defect models across animal species.

[Impact of the Lubrizol Rouen plant fire of September 26, 2019 on ophthalmic emergency room visits]. / Impact de l'incendie de l'usine Lubrizol à Rouen le 26 septembre 2019 sur la fréquentation des urgences ophtalmologiques.

INTRODUCTION: A fire at the Lubrizol chemical factory in Rouen on September 26, 2019 generated a huge column of smoke directed northeast toward the city. As the eye might be particularly affected by the smoke and other toxic emissions from the fire, we assessed the impact of this industrial and ecological disaster on irritative eye surface disease in the week following the accident. MATERIALS AND METHODS: We retrospectively collected the medical data of the patients who presented to the Ophthalmology Emergency Department (OED) of Rouen University Hospital (the only OED open during the days following the accident) during the week following the fire (W1). We compared these data with those of patients who presented during the week before the fire (W-1). We also collected data on patients presenting to the ED in general during W-1 and W1, including the number of visits directly related to the fire. RESULTS: 361 patients presented to the OED during W1 following the fire, compared with 384 in W-1. Of these patients, 83 (23%) had ocular surface disease in W1, versus 76 (20%) in W-1. Conjunctivitis was found in 54 patients in W1 (39 viral, 9 allergic, 6 undetermined) versus 44 in W-1 (27 viral, 12 allergic, 5 undetermined). A dry irritative syndrome was present in 29 patients in W1 versus 32 in W-1. Only 4 patients directly attributed their symptoms to the fire: 2 viral conjunctivitis, 1 allergic conjunctivitis and 1 worried patient (at D2, D5, D7 and D7 following the fire respectively). DISCUSSION: The number of emergency eye consultations did not change in the week following the Lubrizol factory fire (except for a decrease the day of the accident, related to the lock-down). There was a higher number of consultations in W1 for conjunctivitis, mostly viral in appearance and probably not directly related to the fire. The number of consultations for dry irritative syndrome was comparable between the two periods. Despite major media coverage of the event at the national level and a very high level of concern among the population, the fire does not seem to have had an effect on OED activity at Rouen University Hospital, nor on general ED visits. The stay-at-home order on the first day may have had a protective effect, avoiding direct exposure to smoke. The long-term consequences of the soot deposits on the ground as the smoke cloud passed over remain undetermined and are under surveillance. A review of the literature on the ocular consequences of industrial accidents is presented. CONCLUSION: The Ophthalmology Emergency Department did not record increased activity in the week following the Lubrizol Rouen fire, and ocular surface disease did not give rise to more consultations than the week before the fire. This suggests that there was no or minimal immediate ocular toxicity of the smoke from the fire.

Bone morphogenetic proteins for articular cartilage regeneration.

Degeneration of articular cartilage (AC) tissue is the most common cause of osteoarthritis (OA) and rheumatoid arthritis. Bone morphogenetic proteins (BMPs) play important roles in bone and cartilage formation. This article reviews the experimental and clinical applications of BMPs in cartilage regeneration. Experimental evidence indicates that BMPs play an important role in protection against cartilage damage caused by inflammation or trauma, by binding to different receptor combinations and, consequently, activating different intracellular signaling pathways. Loss of function of BMP-related receptors contributes to the decreased intrinsic repair capacity of damaged cartilage and, thus, the multifunctional effects of BMPs make them attractive tools for the treatment of cartilage damage in patients with degenerative diseases. However, the development of BMP therapy as a treatment modality for cartilage regeneration has been hampered by certain factors, such as the eligibility of participants in clinical trials, financial support, drug delivery carrier safety, availabilities of effective scaffolds, appropriate selection of optimal dose and timing of administration, and side effects. Further research is needed to overcome these issues for future routine clinical applications. Research and development leading to the successful application of BMPs can initiate a new era in the treatment of cartilage degenerative diseases like OA.

αv integrins on mesenchymal cells regulate skeletal and cardiac muscle fibrosis.

Mesenchymal cells expressing platelet-derived growth factor receptor beta (PDGFRß) are known to be important in fibrosis of organs such as the liver and kidney. Here we show that PDGFRß+ cells contribute to skeletal muscle and cardiac fibrosis via a mechanism that depends on αv integrins. Mice in which αv integrin is depleted in PDGFRß+ cells are protected from cardiotoxin and laceration-induced skeletal muscle fibrosis and angiotensin II-induced cardiac fibrosis. In addition, a small-molecule inhibitor of αv integrins attenuates fibrosis, even when pre-established, in both skeletal and cardiac muscle, and improves skeletal muscle function. αv integrin blockade also reduces TGFß activation in primary human skeletal muscle and cardiac PDGFRß+ cells, suggesting that αv integrin inhibitors may be effective for the treatment and prevention of a broad range of muscle fibroses.

Evolving paradigms in clinical pharmacology and therapeutics for the treatment of Duchenne muscular dystrophy.

Progressive muscle weakness and degeneration due to the lack of dystrophin eventually leads to the loss of independent ambulation by the middle of the patient's second decade, and a fatal outcome due to cardiac or respiratory failure by the third decade. More specifically, loss of sarcolemmal dystrophin and the dystrophin-associated glycoprotein (DAG) complex promotes muscle fiber damage during muscle contraction. This process results in an efflux of creatine kinase (CK), an influx of calcium ions, and the recruitment of T cells, macrophages, and mast cells to the damaged muscle, causing progressive myofiber necrosis. For the last 20 years, the major goal in the development of therapeutic approaches to alleviate muscle weakness in DMD has been centered on the restoration of dystrophin or proteins that are analogous to dystrophin, such as utrophin, through a variety of modalities including cell therapy, gene therapy, gene correction, and the highly promising techniques utilizing CRISPR/Cas9 technology. Despite the development of new therapeutic options, there still exist numerous challenges that we must face with regard to these new strategies and, consequently, we still do not have any feasible options available to ultimately slow the progression of this devastating disease. The purpose of this article is to highlight the current knowledge and advancements in the evolving paradigms in clinical pharmacology and therapeutics for this devastating musculoskeletal disease.

NF-κB inhibition reveals a novel role for HGF during skeletal muscle repair.

The transcription factor nuclear factor κB (NF-κB)/p65 is the master regulator of inflammation in Duchenne muscular dystrophy (DMD). Disease severity is reduced by NF-κB inhibition in the mdx mouse, a murine DMD model; however, therapeutic targeting of NF-κB remains problematic for patients because of its fundamental role in immunity. In this investigation, we found that the therapeutic effect of NF-κB blockade requires hepatocyte growth factor (HGF) production by myogenic cells. We found that deleting one allele of the NF-κB subunit p65 (p65+/-) improved the survival and enhanced the anti-inflammatory capacity of muscle-derived stem cells (MDSCs) following intramuscular transplantation. Factors secreted from p65+/- MDSCs in cell cultures modulated macrophage cytokine expression in an HGF-receptor-dependent manner. Indeed, we found that following genetic or pharmacologic inhibition of basal NF-κB/p65 activity, HGF gene transcription was induced in MDSCs. We investigated the role of HGF in anti-NF-κB therapy in vivo using mdx;p65+/- mice, and found that accelerated regeneration coincided with HGF upregulation in the skeletal muscle. This anti-NF-κB-mediated dystrophic phenotype was reversed by blocking de novo HGF production by myogenic cells following disease onset. HGF silencing resulted in increased inflammation and extensive necrosis of the diaphragm muscle. Proteolytic processing of matrix-associated HGF is known to activate muscle stem cells at the earliest stages of repair, but our results indicate that the production of a second pool of HGF by myogenic cells, negatively regulated by NF-κB/p65, is crucial for inflammation resolution and the completion of repair in dystrophic skeletal muscle. Our findings warrant further investigation into the potential of HGF mimetics for the treatment of DMD.

Automated classification and visualization of fluorescent live cell microscopy images.

Robotic, high-throughput microscopy is a powerful tool for small molecule screening and classifying cell phenotype, proteomic and genomic data. An important hurdle in the field is the automated classification and visualization of results collected from a data set of tens of thousands of images. We present a method that approaches these problems from the perspective of flow cytometry with supporting open-source code. Image analysis software was created that allowed high-throughput microscopy data to be analysed in a similar manner as flow cytometry. Each cell on an image is considered an object and a series of gates similar to flow cytometry is used to classify and quantify the properties of cells including size and level of fluorescent intensity. This method is released with open-source software and code that demonstrates the method's implementation. Accuracy of the software was determined by measuring the levels of apoptosis in a primary murine myoblast cell line after exposure to staurosporine and comparing these results to flow cytometry.

The effect of platelet-rich plasma on the regenerative therapy of muscle derived stem cells for articular cartilage repair.

OBJECTIVE: Platelet-rich plasma (PRP) is reported to promote collagen synthesis and cell proliferation as well as enhance cartilage repair. Our previous study revealed that the intracapsular injection of muscle derived stem cells (MDSCs) expressing bone morphogenetic protein 4 (BMP-4) combined with soluble Flt-1 (sFlt1) was effective for repairing articular cartilage (AC) after osteoarthritis (OA) induction. The current study was undertaken to investigate whether PRP could further enhance the therapeutic effect of MDSC therapy for the OA treatment. METHODS: MDSCs expressing BMP-4 and sFlt1 were mixed with PRP and injected into the knees of immunodeficient rats with chemically induced OA. Histological assessments were performed 4 and 12 weeks after cell transplantation. Moreover, to elucidate the repair mechanisms, we performed in vitro assays to assess cell proliferation, adhesion, migration and mixed pellet co-culture of MDSCs and OA chondrocytes. RESULTS: The addition of PRP to MDSCs expressing BMP-4 and sFlt1 significantly improved AC repair histologically at week 4 compared to MDSCs expressing BMP-4 and sFlt1 alone. Higher numbers of cells producing type II collagen and lower levels of chondrocyte apoptosis were observed by MDSCs expressing BMP-4 and sFlt1 and mixed with PRP. In the in vitro experiments, the addition of PRP promoted proliferation, adhesion and migration of the MDSCs. During chondrogenic pellet culture, PRP tended to increase the number of type II collagen producing cells and in contrast to the in vivo data, it increased cell apoptosis. CONCLUSIONS: Our findings indicate that PRP can promote the therapeutic potential of MDSCs expressing BMP-4 and sFlt1 for AC repair (4 weeks post-treatment) by promoting collagen synthesis, suppressing chondrocyte apoptosis and finally by enhancing the integration of the transplanted cells in the repair process.

AAV-based shRNA silencing of NF-κB ameliorates muscle pathologies in mdx mice.

Chronic inflammation, promoted by an upregulated NF-kappa B (NF-κB) pathway, has a key role in Duchenne muscular dystrophy (DMD) patients' pathogenesis. Blocking the NF-κB pathway has been shown to be a viable approach to diminish chronic inflammation and necrosis in the dystrophin-defective mdx mouse, a murine DMD model. In this study, we used the recombinant adeno-associated virus serotype 9 (AAV9) carrying an short hairpin RNA (shRNA) specifically targeting the messenger RNA of NF-κB/p65 (p65-shRNA), the major subunit of NF-κB associated with chronic inflammation in mdx mice. We examined whether i.m. AAV9-mediated delivery of p65-shRNA could decrease NF-κB activation, allowing for amelioration of muscle pathologies in 1- and 4-month-old mdx mice. At 1 month after treatment, NF-κB/p65 levels were significantly decreased by AAV gene transfer of p65-shRNA in the two ages of treatment groups, with necrosis significantly decreased compared with controls. Quantitative analysis revealed that central nucleation (CN) of the myofibers of p65-shRNA-treated 1-month-old mdx muscles was reduced from 67 to 34%, but the level of CN was not significantly decreased in treated 4-month-old mdx mice. Moreover, delivery of the p65-shRNA enhanced the capacity of myofiber regeneration in old mdx mice treated at 4 months of age when the dystrophic myofibers were most exhausted; however, such p65 silencing diminished the myofiber regeneration in young mdx mice treated at 1 month of age. Taken together, these findings demonstrate that the AAV-mediated delivery of p65-shRNA has the capacity to ameliorate muscle pathologies in mdx mice by selectively reducing NF-κB/p65 activity.

Inhibition of the IKK/NF-κB pathway by AAV gene transfer improves muscle regeneration in older mdx mice.

The IκB kinase (IKKα, ß and the regulatory subunit IKKγ) complex regulates nuclear factor of κB (NF-κB) transcriptional activity, which is upregulated in many chronic inflammatory diseases. NF-κB signaling promotes inflammation and limits muscle regeneration in Duchenne muscular dystrophy (DMD), resulting in fibrotic and fatty tissue replacement of muscle that exacerbates the wasting process in dystrophic muscles. Here, we examined whether dominant-negative forms of IKKα (IKKα-dn) and IKKß (IKKß-dn) delivered by adeno-associated viral (AAV) vectors to the gastrocnemius (GAS) and tibialis anterior (TA) muscles of 1, 2 and 11-month-old mdx mice, a murine DMD model, block NF-κB activation and increase muscle regeneration. At 1 month post-treatment, the levels of nuclear NF-κB in locally treated muscle were decreased by gene transfer with either AAV-CMV-IKKα-dn or AAV-CMV-IKKß-dn, but not by IKK wild-type controls (IKKα and ß) or phosphate-buffered saline (PBS). Although treatment with AAV-IKKα-dn or AAV-IKKß-dn vectors had no significant effect on muscle regeneration in young mdx mice treated at 1 and 2 months of age and collected 1 month later, treatment of old (11 months) mdx with AAV-CMV-IKKα-dn or AAV-CMV-IKKß-dn significantly increased levels of muscle regeneration. In addition, there was a significant decrease in myofiber necrosis in the AAV-IKKα-dn- and AAV-IKKß-dn-treated mdx muscle in both young and old mice. These results demonstrate that inhibition of IKKα or IKKß in dystrophic muscle reduces the adverse effects of NF-κB signaling, resulting in a therapeutic effect. Moreover, these results clearly demonstrate the therapeutic benefits of inhibiting NF-κB activation by AAV gene transfer in dystrophic muscle to promote regeneration, particularly in older mdx mice, and block necrosis.

Isolation of myogenic progenitor populations from Pax7-deficient skeletal muscle based on adhesion characteristics.

In an attempt to determine whether muscle-derived stem cells are distinct from satellite cells, we investigated whether muscle-derived stem cells could be isolated from the skeletal muscle of Pax7-deficient mice, which have been shown to be devoid of or to contain only a minimal number of satellite cells. Utilizing a technique that separates cells based on their adhesion characteristics (the preplate technique), several distinct populations of muscle-derived cells were isolated. In these mice, the Pax7 gene was knocked out with the insertion of the LacZ gene. One population was both rapidly adhering, LacZ-positive, and displayed a high myogenic index, but was rapidly lost to terminal differentiation when continuously replated. A second population, which persisted over 50 passages, was LacZ-negative and displayed a low myogenic index. Although Pax3 may have acted as a compensatory mechanism for the myogenic commitment of the LacZ-positive cells, the LacZ-negative cells, despite expressing Pax3, required Pax7 transduction to restore their myogenic capacity. We believe that these two populations of myogenic progenitor cells, each endowed with different adhesion characteristics, may help explain the discrepancy in the literature concerning the presence of myogenic cells found in Pax7-deficient mice.

1-year follow-up of autologous muscle-derived stem cell injection pilot study to treat stress urinary incontinence.

We hereby report a 1-year follow-up on eight women in the first North America trial in which stress urinary incontinence (SUI) was treated with muscle-derived stem cell injections. Mean and median follow-up in this group was 16.5 and 17 months (range 3-24 months). Improvement in SUI was seen in five of eight women, with one achieving total continence. Onset of improvement was between 3 and 8 months after injection. Cure or improvement continued at a median of 10 months. No serious adverse events were reported.

Review: gene- and stem cell-based therapeutics for bone regeneration and repair.

Many clinical conditions require regeneration or implantation of bone. This is one focus shared by neurosurgery and orthopedics. Current therapeutic options (bone grafting and protein-based therapy) do not provide satisfying solutions to the problem of massive bone defects. In the past few years, gene- and stem cell-based therapy has been extensively studied to achieve a viable alternative to current solutions offered by modern medicine for bone-loss repair. The use of adult stem cells for bone regeneration has gained much focus. This unique population of multipotential cells has been isolated from various sources, including bone marrow, adipose, and muscle tissues. Genetic engineering of adult stem cells with potent osteogenic genes has led to fracture repair and rapid bone formation in vivo. It is hypothesized that these genetically modified cells exert both an autocrine and a paracrine effects on host stem cells, leading to an enhanced osteogenic effect. The use of direct gene delivery has also shown much promise for in vivo bone repair. Several viral and nonviral methods have been used to achieve substantial bone tissue formation in various sites in animal models. To advance these platforms to the clinical setting, it will be mandatory to overcome specific hurdles, such as control over transgene expression, viral vector toxicity, and prolonged culture periods of therapeutic stem cells. This review covers a prospect of cell and gene therapy for bone repair as well as some very recent advancements in stem cell isolation, genetic engineering, and exogenous control of transgene expression.

Injection of skeletal muscle-derived cells into the penis improves erectile function.

We investigated the effect of intrapenile injection of muscle-derived cells (MDC) on the erectile function in rats with bilateral cavernous nerve injury. Rat MDC were harvested and transduced with a retrovirus expressing the lacZ gene. Hanks' balanced salt solution (HBSS) (20 microl) or MDC (1 x 10(6) cells/side) were injected in each corpora cavernosa immediately before bilateral cavernous nerve transection. Intracavernous pressures (ICP) were measured 2 or 4 weeks after surgery with electrical stimulation of the pelvic nerves. Mean maximal ICP of sham group was significantly lower than that of control group both at 2 and 4 weeks after surgery. When MDC were injected into the penis, ICP improved over the sham-injected group at both 2 and 4 weeks after surgery. Percent area of PGP 9.5 staining was significantly greater in MDC-injected penis than in sham-injected at 2 and 4 weeks. Penile MDC injection can facilitate recovery of injured penile innervation and improve erectile function.

The use of relaxin improves healing in injured muscle.

To improve the functional recovery of injured skeletal muscle, we have focused our efforts on both enhancement of muscle regeneration and prevention of fibrosis. The polypeptide cytokine/growth factor relaxin can inhibit fibrous tissue formation in many tissues. As a member of the insulin-like growth factor family, relaxin also is a potential stimulator of muscle regeneration. In the current experiment, we examined the antifibrotic effect of relaxin in injured skeletal muscle. We also investigated if the injection of relaxin would influence muscle regeneration after injury. Our results demonstrate that relaxin treatment improved histologic and physiologic healing of muscles subjected to traumatic injury.

Long-term self-renewal of postnatal muscle-derived stem cells.

The ability to undergo self-renewal is a defining characteristic of stem cells. Self-replenishing activity sustains tissue homeostasis and regeneration. In addition, stem cell therapy strategies require a heightened understanding of the basis of the self-renewal process to enable researchers and clinicians to obtain sufficient numbers of undifferentiated stem cells for cell and gene therapy. Here, we used postnatal muscle-derived stem cells to test the basic biological assumption of unlimited stem cell replication. Muscle-derived stem cells (MDSCs) expanded for 300 population doublings (PDs) showed no indication of replicative senescence. MDSCs preserved their phenotype (ScaI+/CD34+/desmin(low)) for 200 PDs and were capable of serial transplantation into the skeletal muscle of mdx mice, which model Duchenne muscular dystrophy. MDSCs expanded to this level exhibited high skeletal muscle regeneration comparable with that exhibited by minimally expanded cells. Expansion beyond 200 PDs resulted in lower muscle regeneration, loss of CD34 expression, loss of myogenic activity, and increased growth on soft agar, suggestive of inevitable cell aging attributable to expansion and possible transformation of the MDSCs. Although these results raise questions as to whether cellular transformations derive from cell culturing or provide evidence of cancer stem cells, they establish the remarkable long-term self-renewal and regeneration capacity of postnatal MDSCs.

Regeneration of dystrophin-expressing myocytes in the mdx heart by skeletal muscle stem cells.

Cell transplantation holds promise as a potential treatment for cardiac dysfunction. Our group has isolated populations of murine skeletal muscle-derived stem cells (MDSCs) that exhibit stem cell-like properties. Here, we investigated the fate of MDSCs after transplantation into the hearts of dystrophin-deficient mdx mice, which model Duchenne muscular dystrophy (DMD). Transplanted MDSCs generated large grafts consisting primarily of numerous dystrophin-positive myocytes and, to a lesser degree, dystrophin-negative non-myocytes that expressed an endothelial phenotype. Most of the dystrophin-positive myocytes expressed a skeletal muscle phenotype and did not express a cardiac phenotype. However, some donor myocytes, located at the graft-host myocardium border, were observed to express cardiac-specific markers. More than half of these donor cells that exhibited a cardiac phenotype still maintained a skeletal muscle phenotype, demonstrating a hybrid state. Sex-mismatched donors and hosts revealed that many donor-derived cells that acquired a cardiac phenotype did so through fusion with host cardiomyocytes. Connexin43 gap junctions were not expressed by donor-derived myocytes in the graft. Scar tissue formation in the border region may inhibit the fusion and gap junction connections between donor and host cells. This study demonstrates that MDSC transplantation warrants further investigation as a potential therapy for cardiac dysfunction in DMD.

[Ex-vivo gene therapy with BMP-4 for critically sized defects and enhancement of fracture healing in an osteoporotic animal model]. / Ex-vivo-Gentherapie mit BMP4 bei kritischen Substanzdefekten und Frakturen im osteoporotischen Tiermodell.

Fractures in osteoporotic bones or segment defects are problematic bone lesions with a reduced biological capability of regeneration. We tested the hypothesis that cell-mediated ex vivo gene therapy to deliver BMP4 can heal critically sized defects and improve bone healing in osteoporotic rats. Primary muscle-derived cells were isolated from the hindlimb muscle of rats and retrovirally transduced to express bone morphogenic protein 4 (BMP4). The bone formation was evaluated following local application of these cells in critically sized defects and in fractures of osteoporotic bones. Radiographic analysis revealed bridging callus formation in a critically sized defect in all specimens using muscle-derived cells expressing BMP4 at 12 weeks. These findings were confirmed by histological evaluation, which revealed callus bone formation with good integration to the distal and proximal bone. Following treatment with muscle-derived cells expressing BMP4, the bone healing process in the osteoporotic bone was improved to the level similar to that of normal bone. The ex vivo gene therapy could be a promising tool for the treatment of osteoporotic fractures and critically sized defects. The reduced number of complications (nonunions, loss of reduction, and fragment dislocation), shortening of hospitalization period, and improvement of bone strength are decisive advocates for this treatment option.