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Acute injury of skeletal muscle disrupts myofibres, microvessels and motor innervation. Myofibre regeneration is well characterized, however its relationship with the regeneration of microvessels and motor nerves is undefined. Endothelial cell (EC) ephrin‐B2 (Efnb2) is required for angiogenesis during embryonic development and promotes neurovascular regeneration in the adult. We hypothesized that, following acute injury to skeletal muscle, loss of EC Efnb2 would impair microvascular regeneration and the recovery of neuromuscular junction (NMJ) integrity. Mice (aged 3–6 months) were bred for EC‐specific conditional knockout (CKO) of Efnb2 following tamoxifen injection with non‐injected CKO mice as controls (CON). The gluteus maximus, tibialis anterior or extensor digitorum longus muscle was then injured with local injection of BaCl2. Intravascular staining with wheat germ agglutinin revealed diminished capillary area in the gluteus maximus of CKO vs. CON at 5 days post‐injury (dpi); both recovered to uninjured (0 dpi) level by 10 dpi. At 0 dpi, tibialis anterior isometric force of CKO was less than CON. At 10 dpi, isometric force was reduced by half in both groups. During intermittent contractions (75 Hz, 330 ms s–1, 120 s), isometric force fell during indirect (sciatic nerve) stimulation whereas force was maintained during direct (electrical field) stimulation of myofibres. Neuromuscular transmission failure correlated with perturbed presynaptic (terminal Schwann cells) and postsynaptic (nicotinic acetylcholine receptors) NMJ morphology in CKO. Resident satellite cell number on extensor digitorum longus myofibres did not differ between groups. Following acute injury of skeletal muscle, loss of Efnb2 in ECs delays capillary regeneration and attenuates recovery of NMJ structure and function. Key points: The relationship between microvascular regeneration and motor nerve regeneration following skeletal muscle injury is undefined. Expression of Efnb2 in endothelial cells (ECs) is essential to vascular development and promotes neurovascular regeneration in the adult.To test the hypothesis that EfnB2 in ECs is required for microvascular regeneration and myofibre reinnervation, we induced conditional knockout of Efnb2 in ECs of mice. Acute injury was then induced by BaCl2 injection into gluteus maximus, tibialis anterior or extensor digitorum longus (EDL) muscle.Capillary regeneration was reduced at 5 days post‐injury (dpi) in gluteus maximus of conditional knockout vs. controls; at 10 dpi, neither differed from uninjured. Nerve stimulation revealed neuromuscular transmission failure in tibialis anterior with perturbed neuromuscular junction structure. Resident satellite cell number on EDL myofibres did not differ between groups.Conditional knockout of EC Efnb2 delays capillary regeneration and attenuates recovery of neuromuscular junction structure and function. [ABSTRACT FROM AUTHOR]

Currently the standard surgical treatment for bladder defects is augmentation cystoplasty with autologous tissues, which has many side effects. Biomaterials such as small intestine submucosa (SIS) can provide an alternative scaffold for the repair as bladder patches. Previous studies have shown that SIS could enhance the capacity and compliance of the bladder, but its application is hindered by issues like limited smooth muscle regeneration and stone formation since the fast degradation and poor mechanical properties of the SIS. Procyanidins (PC), a natural bio-crosslinking agent, has shown anti-calcification, anti-inflammatory and anti-oxidation properties. More importantly, PC and SIS can crosslink through hydrogen bonds, which may endow the material with enhanced mechanical property and stabilized functionalities. In this study, various concentrations of PC-crosslinked SIS (PC-SIS) were prepared to repair the full-thickness bladder defects, with an aim to reduce complications and enhance bladder functions. In vitro assays showed that the crosslinking has conferred the biomaterial with superior mechanical property and anti-calcification property, ability to promote smooth muscle cell adhesion and upregulate functional genes expression. Using a rabbit model with bladder defects, we demonstrated that the PC-SIS scaffold can rapidly promote in situ tissue regrowth and regeneration, in particular smooth muscle remodeling and improvement of urinary functions. The PC-SIS scaffold has therefore provided a promising material for the reconstruction of a functional bladder.

Abstract Background Degeneration of smooth muscles in sphincters can cause debilitating diseases such as fecal incontinence. Skeletal muscle-derived cells have been effectively used in clinics for the regeneration of the skeletal muscle sphincters, such as the external anal or urinary sphincter. However, little is known about the in vitro smooth muscle differentiation potential and in vivo regenerative potential of skeletal muscle-derived cells. Methods Myogenic progenitor cells (MPC) were isolated from the skeletal muscle and analyzed by flow cytometry and in vitro differentiation assays. The differentiation of MPC to smooth muscle cells (MPC-SMC) was evaluated by immunofluorescence, flow cytometry, patch-clamp, collagen contraction, and microarray gene expression analysis. In vivo engraftment of MPC-SMC was monitored by transplanting reporter protein-expressing cells into the pyloric sphincter of immunodeficient mice. Results MPC derived from human skeletal muscle expressed mesenchymal surface markers and exhibit skeletal myogenic differentiation potential in vitro. In contrast, they lack hematopoietic surface marker, as well as adipogenic, osteogenic, and chondrogenic differentiation potential in vitro. Cultivation of MPC in smooth muscle differentiation medium significantly increases the fraction of alpha smooth muscle actin (aSMA) and smoothelin-positive cells, while leaving the number of desmin-positive cells unchanged. Smooth muscle-differentiated MPC (MPC-SMC) exhibit increased expression of smooth muscle-related genes, significantly enhanced numbers of CD146- and CD49a-positive cells, and in vitro contractility and express functional Cav and Kv channels. MPC to MPC-SMC differentiation was also accompanied by a reduction in their skeletal muscle differentiation potential. Upon removal of the smooth muscle differentiation medium, a major fraction of MPC-SMC remained positive for aSMA, suggesting the definitive acquisition of their phenotype. Transplantation of murine MPC-SMC into the mouse pyloric sphincter revealed engraftment of MPC-SMC based on aSMA protein expression within the host smooth muscle tissue. Conclusions Our work confirms the ability of MPC to give rise to smooth muscle cells (MPC-SMC) with a well-defined and stable phenotype. Moreover, the engraftment of in vitro-differentiated murine MPC-SMC into the pyloric sphincter in vivo underscores the potential of this cell population as a novel cell therapeutic treatment for smooth muscle regeneration of sphincters.

Background: Degeneration of smooth muscles in sphincters can cause debilitating diseases such as fecal incontinence. Skeletal muscle-derived cells have been effectively used in clinics for the regeneration of the skeletal muscle sphincters, such as the external anal or urinary sphincter. However, little is known about the in vitro smooth muscle differentiation potential and in vivo regenerative potential of skeletal muscle-derived cells. Methods: Myogenic progenitor cells (MPC) were isolated from the skeletal muscle and analyzed by flow cytometry and in vitro differentiation assays. The differentiation of MPC to smooth muscle cells (MPC-SMC) was evaluated by immunofluorescence, flow cytometry, patch-clamp, collagen contraction, and microarray gene expression analysis. In vivo engraftment of MPC-SMC was monitored by transplanting reporter protein-expressing cells into the pyloric sphincter of immunodeficient mice. Results: MPC derived from human skeletal muscle expressed mesenchymal surface markers and exhibit skeletal myogenic differentiation potential in vitro. In contrast, they lack hematopoietic surface marker, as well as adipogenic, osteogenic, and chondrogenic differentiation potential in vitro. Cultivation of MPC in smooth muscle differentiation medium significantly increases the fraction of alpha smooth muscle actin (aSMA) and smoothelin-positive cells, while leaving the number of desmin-positive cells unchanged. Smooth muscle-differentiated MPC (MPC-SMC) exhibit increased expression of smooth muscle-related genes, significantly enhanced numbers of CD146- and CD49a-positive cells, and in vitro contractility and express functional Cav and Kv channels. MPC to MPC-SMC differentiation was also accompanied by a reduction in their skeletal muscle differentiation potential. Upon removal of the smooth muscle differentiation medium, a major fraction of MPC-SMC remained positive for aSMA, suggesting the definitive acquisition of their phenotype. Transplantation of murine MPC-SMC into the mouse pyloric sphincter revealed engraftment of MPC-SMC based on aSMA protein expression within the host smooth muscle tissue. Conclusions: Our work confirms the ability of MPC to give rise to smooth muscle cells (MPC-SMC) with a well-defined and stable phenotype. Moreover, the engraftment of in vitro-differentiated murine MPC-SMC into the pyloric sphincter in vivo underscores the potential of this cell population as a novel cell therapeutic treatment for smooth muscle regeneration of sphincters. [ABSTRACT FROM AUTHOR]

Vaginal reconstruction is the main solution to the problem of sexuality and gender roles for patients with no vagina. A tissue‐engineered vagina may be the best choice. However, many defects have been found in neovaginas reconstructed with a graft only. In this study, we investigated whether a stem cell‐seeded graft would accelerate the morphological and functional recovery of neovaginas. CM‐DiI‐labeled bone marrow mesenchymal stem cell (MSC)‐seeded small intestinal submucosa (SIS) (SIS+MSCs group) was used for vaginal reconstruction in a rat model; unseeded SIS (SIS group) was used as a control. The neovaginas of each group were harvested at 4 and 12 weeks after surgery. Morphological analyses were performed using hematoxylin and eosin (H&E) staining and immunohistochemical staining for α‐smooth muscle actin (SMA), protein gene product 9.5(PGP9.5), and CD34. Functional recovery was evaluated using an organ bath study. The role of MSCs in the neovagina was analyzed by immunofluorescence and molecular biology methods. At the 4th week, a regenerated epithelium covered the whole neovagina in both groups. A small amount of smooth muscle regeneration was found in the neovagina. Up to the 12th week, nerve fibers appeared. There were more smooth muscle and nerve fibers, along with better contractility, in the neovagina of the SIS+MSCs group. Further study showed that the MSCs differentiated into smooth muscles at the 4th week. A higher microvessel density (MVD) and more vascular endothelial growth factor (VEGF) were found in the neovagina of the SIS+MSCs group. In short, MSCs accelerate the structural and functional recovery of the neovagina. [ABSTRACT FROM AUTHOR]

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Vascular smooth muscle cells (VSMCs) play essential roles in regulating blood vessel form and function. Regeneration of functional vascular smooth muscle tissue to repair vascular diseases is an area of intense research in tissue engineering and regenerative medicine. For functional vascular smooth muscle tissue regeneration to become a practical therapy over the next decade, the field will need to have access to VSMC sources that are effective, robust and safe. While pluripotent stem cells hold good future promise to this end, more immediate translation is expected to come from approaches that generate functional VSMCs from adult sources of multipotent adipose-derived and bone marrow-derived mesenchymal stromal cells (ASCs and BMSCs). The research to this end is extensive and is dominated by studies relating to classical biochemical signalling molecules used to induce differentiation of ASCs and BMSCs. However, prolonged use of the biochemical induction factors is costly and can cause potential endotoxin contamination in the culture. Over recent years several non-traditional differentiation approaches have been devised to mimic defined aspects of the native micro-environment in which VSMCs reside to contribute to the differentiation of VSMC-like cells from ASCs and BMSCs. In this review, the promises and limitations of several non-traditional culture approaches (e.g., co-culture, biomechanical, and biomaterial stimuli) targeting VSMC differentiation are discussed. The extensive crosstalk between the underlying signalling cascades are delineated and put into a translational context. It is expected that this review will not only provide significant insight into VSMC differentiation strategies for vascular smooth muscle tissue engineering applications, but will also highlight the fundamental importance of engineering the cellular microenvironment on multiple scales (with consideration of different combinatorial pathways) in order to direct cell differentiation fate and obtain cells of a desired and stable phenotype. These strategies may ultimately be applied to different sources of stem cells in the future for a range of biomaterial and tissue engineering disciplines. [ABSTRACT FROM AUTHOR]

We have demonstrated previously that peripheral blood monocytes can be converted in vitro to a multipotent stem cell-like cell termed programmable cell of monocytic origin (PCMO) and subsequently into cells with chondrocyte-like phenotype. Here, we investigated whether PCMO could also be differentiated into osteoblast-like cells using growth factors with known osteoinductive potency. Following stimulation with BMP-2, BMP-7, IGF-1 or TGF-β1 for 7 and 14 days, PCMOs were analyzed for mRNA expression of collagen types I and V, alkaline phosphatase, osteocalcin, runt-related transcription factor-2 (Runx2) and Osterix (Osx) by quantitative RT-PCR (qPCR) and the levels of collagen I in culture supernatants by ELISA. The expression of osteoblastic markers was evident, albeit at a different extent in cultures of PCMOs after treatment with the above-mentioned growth factors. Culture supernatants from PCMOs stimulated for 6–10 days with BMP-2, BMP-7, IGF-1 or TGF-β1 contained high levels of collagen type I, together with earlier data indicating synthesis and proper secretion. The findings suggest that PCMOs can transform into cells that are phenotypically similar to osteoblasts and identify these cells as osteochondroprogenitors. The possibility of differentiating PCMOs from peripheral blood in sizable quantities could be a novel way to obtain autologous bone-like substitutes without donor-site morbidity. [ABSTRACT FROM AUTHOR]

The secretedWnt signaling molecules are essential to the coordination of cell-fate decision making in multicellular organisms. In adult animals, the secreted Wnt proteins are critical for tissue regeneration and frequently contribute to cancer. Small molecules that disable the Wnt acyltransferase Porcupine (Porcn) are candidate anticancer agents in clinical testing. Here we have systematically assessed the effects of the Porcn inhibitor (WNT-974) on the regeneration of several tissue types to identify potentially unwanted chemical effects that could limit the therapeutic utility of such agents. An unanticipated observation from these studies is proregenerative responses in heart muscle induced by systemic chemical suppression of Wnt signaling. Using in vitro cultures of several cell types found in the heart, we delineate theWnt signaling apparatus supporting an antiregenerative transcriptional program that includes a subunit of the nonfibrillar collagen VI. Similar to observations seen in animals exposed to WNT-974, deletion of the collagen VI subunit, COL6A1, has been shown to decrease aberrant remodeling and fibrosis in infarcted heart tissue. We demonstrate that WNT-974 can improve the recovery of heart function after left anterior descending coronary artery ligation by mitigating adverse remodeling of infarcted tissue. Injured heart tissue exposed to WNT-974 exhibits decreased scarring and reduced Col6 production. Our findings support the development of Porcn inhibitors as antifibrotic agents that could be exploited to promote heart repair following injury. [ABSTRACT FROM AUTHOR]

Skeletal muscle has an exceptional ability to regenerate and adapt following injury. Tissue engineering approaches (e.g. cell therapy, scaffolds, and pharmaceutics) aimed at enhancing or promoting muscle regeneration from severe injuries are a promising and active field of research. Computational models are beginning to advance the field by providing insight into regeneration mechanisms and therapies. In this paper, we summarize the contributions computational models have made to understanding muscle remodeling and the functional implications thereof. Next, we describe a new agent-based computational model of skeletal muscle inflammation and regeneration following acute muscle injury. Our computational model simulates the recruitment and cellular behaviors of key inflammatory cells (e.g. neutrophils and M1 and M2 macrophages) and their interactions with native muscle cells (muscle fibers, satellite stem cells, and fibroblasts) that result in the clearance of necrotic tissue and muscle fiber regeneration. We demonstrate the ability of the model to track key regeneration metrics during both unencumbered regeneration and in the case of impaired macrophage function. We also use the model to simulate regeneration enhancement when muscle is primed with inflammatory cells prior to injury, which is a putative therapeutic intervention that has not yet been investigated experimentally. Computational modeling of muscle regeneration, pursued in combination with experimental analyses, provides a quantitative framework for evaluating and predicting muscle regeneration and enables the rational design of therapeutic strategies for muscle recovery. [ABSTRACT FROM AUTHOR]

Spinal cord injury (SCI) is a devastating condition that leaves patients with limited motor and sensory function at and below the injury site, with little to no hope of a meaningful recovery. Because of their ability to mimic multiple features of central nervous system (CNS) tissues, injectable hydrogels are being developed that can participate as therapeutic agents in reducing secondary injury and in the regeneration of spinal cord tissue. Injectable biomaterials can provide a supportive substrate for tissue regeneration, deliver therapeutic factors, and regulate local tissue physiology. Recent reports of increasing intraspinal pressure after SCI suggest that this physiological change can contribute to injury expansion, also known as secondary injury. Hydrogels contain high water content similar to native tissue, and many hydrogels absorb water and swell after formation. In the case of injectable hydrogels for the spinal cord, this process often occurs in or around the spinal cord tissue, and thus may affect intraspinal pressure. In the future, predictable swelling properties of hydrogels may be leveraged to control intraspinal pressure after injury. Here, we review the physiology of SCI, with special attention to the current clinical and experimental literature, underscoring the importance of controlling intraspinal pressure after SCI. We then discuss how hydrogel fabrication, injection, and swelling can impact intraspinal pressure in the context of developing injectable biomaterials for SCI treatment. [ABSTRACT FROM AUTHOR]

Background: Intra-articular cellular therapy injections constitute an appealing strategy that may modify the intra-articular milieu or regenerate cartilage in the settings of osteoarthritis and focal cartilage defects. However, little consensus exists regarding the indications for cellular therapies, optimal cell sources, methods of preparation and delivery, or means by which outcomes should be reported.Methods: We present a systematic review of the current literature regarding the safety and efficacy of cellular therapy delivered by intra-articular injection in the knee that provided a Level of Evidence of III or higher. A total of 420 papers were screened. Methodological quality was assessed using a modified Coleman methodology score.Results: Only 6 studies (4 Level II and 2 Level III) met the criteria to be included in this review; 3 studies were on treatment of osteoarthritis and 3 were on treatment of focal cartilage defects. These included 4 randomized controlled studies without blinding, 1 prospective cohort study, and 1 retrospective therapeutic case-control study. The studies varied widely with respect to cell sources, cell characterization, adjuvant therapies, and assessment of outcomes. Outcome was reported in a total of 300 knees (124 in the osteoarthritis studies and 176 in the cartilage defect studies). Mean follow-up was 21.0 months (range, 12 to 36 months). All studies reported improved outcomes with intra-articular cellular therapy and no major adverse events. The mean modified Coleman methodology score was 59.1 ± 16 (range, 32 to 82).Conclusions: The studies of intra-articular cellular therapy injections for osteoarthritis and focal cartilage defects in the human knee suggested positive results with respect to clinical improvement and safety. However, the improvement was modest and a placebo effect cannot be disregarded. The overall quality of the literature was poor, and the methodological quality was fair, even among Level-II and III studies. Effective clinical assessment and optimization of injection therapies will demand greater attention to study methodology, including blinding; standardized quantitative methods for cell harvesting, processing, characterization, and delivery; and standardized reporting of clinical and structural outcomes.Level Of Evidence: Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence. [ABSTRACT FROM AUTHOR]

Previous studies have shown that Fibroblast Growth Factors are present in the regenerating tail tissues of lizards where they may stimulate the process of regeneration. The present study is focused on the immunolocalization of FGF8 and FGF10 in the regenerating lizard tail, two signaling proteins of the apical epidermal cup/ridge and mesenchymal blastema sustaining tail and limb regeneration in amphibians and the development of the tail and limbs in vertebrate embryos. Main immunoreactive protein bands at 15–18 kDa for FGF8/10 are detected in the regenerating epidermis and only a band at 30 or 35 kDa in the underlying connective tissues. FGF8 appears particularly localized in cells and nuclei of the apical epidermal peg and of the ependymal ampulla present at the tip of the regenerating tail. FGF10 is also immuno-localized in the apical epidermis but is particularly intensely localized in the mesenchyme of the apical blastema. In accordance with previous studies, the present observations supports the hypothesis that the apical epidermal peg and the ependymal tube with the few regenerated neurons present within it, release FGF8/10 that may contribute to maintenance of cell proliferation in the apical front of the mesenchyme for the growth of the regenerating tail. [ABSTRACT FROM AUTHOR]

An ideal vascular graft should have the ability to guide the regeneration of neovessels with structure and function similar to those of the native blood vessels. Regeneration of vascular smooth muscle cells (VSMCs) with circumferential orientation within the grafts is crucial for functional vascular reconstruction in vivo . To date, designing and fabricating a vascular graft with well-defined geometric cues to facilitate simultaneously VSMCs infiltration and their circumferential alignment remains a great challenge and scarcely reported in vivo . Thus, we have designed a bi-layered vascular graft, of which the internal layer is composed of circumferentially aligned microfibers prepared by wet-spinning and an external layer composed of random nanofibers prepared by electrospinning. While the internal circumferentially aligned microfibers provide topographic guidance for in vivo regeneration of circumferentially aligned VSMCs, the external random nanofibers can offer enhanced mechanical property and prevent bleeding during and after graft implantation. VSMCs infiltration and alignment within the scaffold was then evaluated in vitro and in viv o. Our results demonstrated that the circumferentially oriented VSMCs and longitudinally aligned ECs were successfully regenerated in vivo after the bi-layered vascular grafts were implanted in rat abdominal aorta. No formation of thrombosis or intimal hyperplasia was observed up to 3 month post implantation. Further, the regenerated neoartery exhibited contraction and relaxation property in response to vasoactive agents. This new strategy may bring cell-free small diameter vascular grafts closer to clinical application. [ABSTRACT FROM AUTHOR]

Vascular smooth muscle cells (VSMCs) are subjected to various types of mechanical forces within the vessel wall. Although it is known that VSMCs undergo cell body reorientation in response to mechanical stimulation, how this mechanical stretch is transduced within the cell into biochemical signals causing cytoskeleton reorganization remains unclear. Cofilin, a protein that controls actin dynamics, is activated by Slingshot phosphatase-dependent serine 3 dephosphorylation by redox-dependent mechanisms. Nox4 is a main source of reactive oxygen species (ROS) in the vessel wall that localizes in association with the cytoskeleton. Therefore, we hypothesize that Nox4 mediates redox-dependent activation of cofilin, which is required for cytoskeletal reorganization and cell reorientation after mechanical stimulation. In this study, we found that mechanical stretch stimulates ROS production in VSMCs and that the signaling that leads to cell reorientation requires hydrogen peroxide but not superoxide. Indeed, mechanical stretch induces cofilin activation and stretch-induced cytoskeletal reorganization, and cell reorientation is inhibited in cells where cofilin activity has been downregulated. Importantly, Nox4-deficient cells fail to activate cofilin and to undergo cell reorientation, a phenotype rescued by the expression of a constitutively active cofilin mutant. Our results demonstrate that in VSMCs mechanical stimulation activates cofilin by a Nox4-dependent mechanism and that this pathway is required for cytoskeleton reorganization and cell reorientation. [ABSTRACT FROM AUTHOR]

The objective of this study was to investigate whether intraperitoneal incubation improves the regenerative capacity of bladder acellular matrix grafts (BAMGs) in a rat model of bladder augmentation. After 2 weeks of incubation in the peritoneum of male rats, BAMG flaps with vascular pedicles were harvested for autologous bladder augmentation. As the control, BAMGs were directly used for bladder augmentation without intraperitoneal incubation. Histological analyses of the incubated BAMGs demonstrated extensive cell growth and vasculature in homogeneous collagen bundles. The cells were positive for vimentin and negative for α-smooth muscle actin and pan-cytokeratin AE1/AE3. Cystography revealed smoother contours of the augmented bladders in the incubated group at 4 and 12 weeks postoperatively. However, the bladder capacity was not significantly different between the two groups. In both groups, the entire urothelium regenerated well without obvious differences. At both time points, compared with the control group, increased numbers of smooth muscle cells (SMCs) and blood vessels were found in the incubated group. At 12 weeks, the SMCs in the incubated group were more similar to those in the native smooth muscle fiber bundles of the bladder. Taken together, our results demonstrated that BAMGs preincubated in the peritoneum promote the regeneration of bladder smooth muscle via neovascularization in a rat bladder augmentation model. [ABSTRACT FROM AUTHOR]

Multipotent perivascular cells (PVCs) have recently gained attention as an alternative source for cell-based regenerative medicine. Because of their rarity in human tissues, the development of efficient methods to isolate and expand PVCs from various fetal and adult tissues is necessary to obtain a clinically relevant number of cells that maintain progenitor potency. We report a simple non-enzymatic isolation (NE) method of PVCs from human umbilical cord (HUC) and compare its efficiency with the conventional collagenase treatment method (CT) in terms of proliferation, immunophenotype, clonogenic capacity, and differentiation potential. Cells isolated by NE expressed the accepted surface marker profile of PVCs and possessed multilineage differentiation potential. Whereas both methods provided similar patterns or levels of immunophenotypes and proliferation, PVCs obtained by NE maintained a higher level of CD146(+) frequency compared with that of CT over passages and displayed greater in vitro osteogenic differentiation potential and clonogenic capacity than CT-PVCs. We assess the potential of various exogenous factors to boost the proliferation of NE- and CT-PVCs in vitro. Supplementation of basic fibroblast growth factor (bFGF) provided optimal conditions that significantly enhanced their proliferation rate. This treatment drove the cells into S phase and increased the proportion of stage-specific antigen-4-positive population without altering other immunophenotypes. Thus, the NE method with bFGF supplementation offers an alternative way for obtaining sufficient numbers of HUCPVCs that have good clonogenic and differentiation potential and that are applicable at therapeutic doses for regenerative medicine. [ABSTRACT FROM AUTHOR]

If one were to compare today's animal growth research to research from a mere 50 yr ago, one would see programs with few similarities. The evolution of this research from whole-animal through cell-based and finally molecular and genomic studies has been enhanced by the identification, isolation, and in vitro evaluation of adipose-and muscle-derived stem cells. This paper will highlight the struggles and the milestones that make this evolving area of research what it is today. The contribution of adipose and muscle stem cell research to development and growth, tissue regeneration, and final carcass composition are reviewed. [ABSTRACT FROM AUTHOR]

Enteric inhibitory neurotransmission is an important feature of the neural regulation of gastrointestinal motility. Purinergic neurotransmission, via P2Y1 receptors, mediates one phase of inhibitory neural control. For decades, ATP has been assumed to be the purinergic neurotransmitter and smooth muscle cells (SMCs) have been considered the primary targets for inhibitory neurotransmission. Recent experiments have cast doubt on both of these assumptions and suggested that another cell type, platelet-derived growth factor receptor-α-positive (PDGFRα+) cells, is the target for purinergic neurotransmission. We compared responses of PDGFRα+ cells and SMCs to several purine compounds to determine if these cells responded in a manner consistent with enteric inhibitory neurotransmission. ATP hyperpolarized PDGFRα+ cells but depolarized SMCs. Only part of the ATP response in PDGFRα+ cells was blocked by MRS 2500, a P2Y1 antagonist. ADP, MRS 2365, β-NAD, and adenosine 5-diphosphateribose, P2Y1 agonists, hyperpolarized PDGFRα+ cells, and these responses were blocked by MRS 2500. Adenosine 5-diphosphateribose was more potent in eliciting hyperpolarization responses than β-NAD. P2Y1 agonists failed to elicit responses in SMCs. Small hyperpolarization responses were elicited in SMCs by a small-conductance Ca2+-activated K+ channel agonist, cyclohexyl-[2-(3,5- dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine, consistent with the low expression and current density of small-conductance Ca2+-activated K+ channels in these cells. Large-amplitude hyperpolarization responses, elicited in PDGFRαα+ cells, but not SMCs, by P2Y1 agonists are consistent with the generation of inhibitory junction potentials in intact muscles in response to purinergic neurotransmission. The responses of PDGFRα+ cells and SMCs to purines suggest that SMCs are unlikely targets for purinergic neurotransmission in colonic muscles. [ABSTRACT FROM AUTHOR]

True tendon regeneration in human patients remains a vision of musculoskeletal therapies. In comparison to other mesenchymal lineages the biology of tenogenic differentiation is barely understood. Specifically, easy and efficient protocols are lacking that might enable tendon cell and tissue differentiation based on adult (stem) cell sources. In the murine mesenchymal progenitor cell line C3H10T½, overexpression of the growth factor bone morphogenetic protein 2 (BMP2) and a constitutively active transcription factor, Smad8 L+MH2, mediates tendon cell differentiation in vitro and the formation of tendon-like tissue in vivo. We hypothesized that during this differentiation secreted factors involved in extracellular matrix formation exert a major impact on tendon development. Gene expression analyses revealed four genes encoding secreted factors that are notably upregulated: periostin, C-type lectin domain family 3 (member b), RNase A4, and follistatin-like 1. These factors have not previously been implicated in tendon biology. Among these, periostin showed a specific expression in tenocytes of adult mouse Achilles tendon and in chondrocytes within the nonmineralized fibrocartilage zone of the enthesis with the calcaneus. Overexpression of periostin alone or in combination with constitutively active BMP receptor type in human mesenchymal stem cells and subsequent implantation into ectopic sites in mice demonstrated a reproducible moderate tenogenic capacity that has not been described before. Therefore, periostin may belong to the factors contributing to the development of tenogenic tissue. [ABSTRACT FROM AUTHOR]

This study identified a distinctive pattern of expression and activity of adenylyl cyclase (AC) and phosphodiesterase (PDE) isoforms in mouse colonic longitudinal smooth muscle cells and determined the changes in their expression and/or activity in response to proinflammatory cytokines (IL-1β and TNF-α) in vitro and 2,4,6 trinitrobenzene sulphonic acid (TNBS)- induced colonic inflammation in vivo. AC5/6 and PDE4D5, expressed in circular muscle cells, were also expressed in longitudinal smooth muscle. cAMP formation was tightly regulated via feedback phosphorylation of AC5/6 and PDE4D5 by PKA. Inhibition of PKA activity by myristoylated PKI blocked phosphorylation of AC5/6 and PDE4D5 and enhanced cAMP formation. TNBS treatment in vivo and IL-1β and TNF-α in vitro induced inducible nitric oxide synthase (iNOS) expression, stimulated ERK1/2 activity, caused iNOS-mediated S-nitrosylation and inhibition of AC5/6, and induced phosphorylation of PDE4D5 and stimulated its activity. The resultant decrease in AC5/6 activity and increase in PDE4D5 activity decreased cAMP formation and smooth muscle relaxation. S-nitrosylation and inhibition of AC5/6 activity were reversed by the iNOS inhibitor 1400W, whereas phosphorylation and activation of PDE4D5 were reversed by the phosphatidylinositol 3-kinase inhibitor LY294002 and the ERK1/2 inhibitor PD98059. The effects of IL-1β or TNF-α on forskolin-stimulated cAMP formation and smooth muscle relaxation reflected inhibition of AC5/6 activity and activation of PDE4D5 and were partly reversed by 1400W or PD98059 and completely reversed by a combination of the two inhibitors. The changes in the cAMP/ PKA signaling and smooth muscle relaxation contribute to colonic dysmotility during inflammation. [ABSTRACT FROM AUTHOR]

Histone deacetylases (HDACs) are a family of enzymes that mediate nucleosomal histone deacetylation and gene expression. Some members of the HDAC family have also been implicated in nonhistone protein deacetylation, which modulates cell-cycle control, differentiation, and cell migration. However, the role of HDACs in smooth muscle contraction is largely unknown. Here, HDAC8 was localized both in the cytoplasm and the nucleus of mouse and human smooth muscle cells. Knockdown of HDAC8 by lentivirus-encoding HDAC8 shRNA inhibited force development in response to acetylcholine. Treatment of smooth muscle tissues with HDAC8 inhibitor XXIV (OSU-HDAC- 44) induced relaxation of precontracted smooth muscle tissues. In addition, cortactin is an actin-regulatory protein that undergoes deacetylation during migration of NIH 3T3 cells. In this study, acetylcholine stimulation induced cortactin deacetylation in mouse and human smooth muscle tissues, as evidenced by immunoblot analysis using antibody against acetylated lysine. Knockdown of HDAC8 by RNAi or treatment with the inhibitor attenuated cortactin deacetylation and actin polymerization without affecting myosin activation. Furthermore, expression of a charge-neutralizing cortactin mutant inhibited contraction and actin dynamics during contractile activation. These results suggest a novel mechanism for the regulation of smooth muscle contraction. In response to contractile stimulation, HDAC8 may mediate cortactin deacetylation, which subsequently promotes actin filament polymerization and smooth muscle contraction. [ABSTRACT FROM AUTHOR]

SUMMARY In this study, we developed a multi-scale finite element (FE) analysis code to obtain the stress and strain that occurred in the smooth muscle cell (SMC) at micro-scale, which was seeded in the real fabricated braid fibril artificial blood vessel. This FE code can predict the dynamic response of stress under the blood pressure loading. We try to establish a computer-aided engineering (CAE)-driven scaffold design technique for the blood vessel regeneration. Until now, there occurred the great progresses for the endothelial cell activation and intima layer regeneration in the blood vessel regeneration study. However, there remains the difficulty of the SMC activation and media layer regeneration. Therefore, many researchers are now studying to elucidate the fundamental mechanism of SMC activation and media layer regeneration by using the biomechanical technique. As the numerical tool, we used the dynamic-explicit FE code PAM-CRASH, ESI Ltd. For the material models, the nonlinear viscoelastic constitutive law was adapted for the human blood vessel, SMC and the extra-cellular matrix, and the elastic law for the polyglycolic acid (PGA) fiber. Through macro-FE and micro-FE analyses of fabricated braid fibril tubes by using PGA fiber under the combined conditions of the orientation angle and the pitch of fiber, we searched an appropriate structure for the stress stimulation for SMC functionalization. Objectives of this study are indicated as follows: to analyze the stress and strain of the human blood vessel and SMC, and, to calculate stress and strain of the real fabricated braid fibril artificial blood vessel and SMC to search an appropriate PGA fiber structure under combined conditions of PGA fiber numbers, 12 and 24, and the helical orientation angles of fiber, 15, 30, 45, 60, and 75 degrees., Finally, we found a braid fibril tube, which has an angle of 15 degree and 12 PGA fibers, as a most appropriate artificial blood vessel for SMC functionalization.Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Muscle regeneration is a complex phenomenon, involving replacement of damaged fibers by new muscle fibers. During this process, there is a tendency to form scar tissue or fibrosis by deposition of collagen that could be detrimental to muscle function. New therapies that could regulate fibrosis and favor muscle regeneration would be important for physical therapy. Low-level laser therapy (LLLT) has been studied for clinical treatment of skeletal muscle injuries and disorders, even though the molecular and cellular mechanisms have not yet been clarified. The aim of this study was to evaluate the effects of LLLT on molecular markers involved in muscle fibrosis and regeneration after cryolesion of the tibialis anterior (TA) muscle in rats. Sixty Wistar rats were randomly divided into three groups: control, injured TA muscle without LLLT, injured TA muscle treated with LLLT. The injured region was irradiated daily for four consecutive days, starting immediately after the lesion using an AlGaAs laser (808 nm, 30 mW, 180 J/cm; 3.8 W/cm, 1.4 J). The animals were sacrificed on the fourth day after injury. LLLT significantly reduced the lesion percentage area in the injured muscle ( p < 0.05), increased mRNA levels of the transcription factors MyoD and myogenin ( p < 0.01) and the pro-angiogenic vascular endothelial growth factor ( p < 0.01). Moreover, LLLT decreased the expression of the profibrotic transforming growth factor TGF-β mRNA ( p < 0.01) and reduced type I collagen deposition ( p < 0.01). These results suggest that LLLT could be an effective therapeutic approach for promoting skeletal muscle regeneration while preventing tissue fibrosis after muscle injury. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: The effects of LLLT were studied during muscle regeneration through gene expression. Methods: It was evaluated 10 and 50J/cm2 doses during 7, 14 and 21days post cryoinjury, through histopathological analysis and mRNA MyoD, Myogenin, Vegf and Cox-2 expression. Results: Irradiated groups presented less inflammatory process than control group after 14 and 21days. Cox-2 levels were downregulated in all irradiated groups after 7, 14 and 21days. On day 7, both treated groups had a downregulation of Vegf levels, and an upregulation after 14 and 21days, mainly with 50J/cm2. The MyoD levels were upregulated with high dose in all periods and with low dose after 21days. Myogenin expression was downregulated in both treated groups after 7days, and was upregulated with 10J/cm2 after 21days. Conclusion: These responses suggest that LLLT can improve the skeletal muscle regeneration through the gene expression stimulation. [Copyright &y& Elsevier]

Myocardin (Mycd), a key factor in smooth muscle cell differentiation, is constitutively located in the nucleus, whereas myocardin- related transcription factorsAand B (MRTF-A/B) reside mostly in the cytoplasm and translocate to the nucleus in a Rhodependent manner. Here, we investigated the nuclear export of Mycd family members. They possess two leucine-rich sequences: L1 in the N terminus and L2 in the Gln-rich domain. Although L2 (but not L1) served as a CRM1-binding site for Mycd, CRM1-mediated nuclear export did not affect its subcellular localization. Serum response factor (SRF) competitively inhibited Mycd/CRM1 interaction. Furthermore, such interaction was autonomously inhibited. The N terminus of Mycd bound intramolecularly to Mycd, resulting in masking L2. In contrast, the CRM1-binding affinity of MRTF-A was much higher than that of Mycd because both L1 and L2 of MRTF-A served as functional CRM1-binding sites, and the autoinhibition observed in the Mycd/CRM1 interaction was absent in the MRTF-A/CRM1 interaction. Additionally, because the SRFbinding affinity of MRTF-A was lower than that of Mycd, the inhibitory effect of SRF on the MRTF-A/CRM1 interaction was weak. Thus, MRTF-A is much more likely to be exported from the nucleus. These differences could be the reason for the distinct subcellular localization of Mycd and MRTF-A. [ABSTRACT FROM AUTHOR]

Objective: One of the major impediments in tissue-engi-neered oral mucosa (TEOM) is the lack of rete ridge (RR) structures that can weaken the connection between the epi-dermis and dermis. This study aimed to investigate the na-tive morphology of RRs as well as the expression of extracel-lular signal-regulated kinase 1/2 (ERK1/2), Ki67, and kera-tin-19, which are related to cell mechanotransduction, proliferation, and sternness in the oral epidermis, respectively. Methods: RR characteristics, including type, density, length, and width, were analyzed in the masticatory mucosa (Mm) and lining mucosa (Lm) sites of 52 specimens. The ex-pression of ERK1/2, Ki67, and keratin-19 was assessed by im-munohistochemistry. ERK1/2 activation by masticatory stim-uli was confirmed in vitro by loading pressure onto cultured keratinocytes isolated from the specimens. Results: Three types of RR were found. The RRs in the Mm and Lm differed. The length and percentage of ERK1/2-positive (%ERK1/2+) basal layer cells had a negative correlation (p = 0.004), where-as the length and %Ki67+ basal layer cells had a positive cor-relation (p = 0.013). The %ERK1/2+ basal layer cells and %ker-atin-19+ basal layer cells had a negative relationship (p =0.011). ERK1/2 activation in the oral epithelium was induced by pressure and propagated in cultured keratinocytes. Conclusion: RRs are longer in the Mm, which may result from the topical basal cell proliferation and migration induced by masticatory pressure via ERK1/2 activation. Our findings pre-liminarily interpret RR histomorphology as influenced by oral masticatory pressure. Results may benefit future studies on RR development and reconstruction in TEOM models to enhance the epidermis-dermis connection. [ABSTRACT FROM AUTHOR]

The periodontal researchers and clinicians, in an effort to develop effective regenerative therapies, have sought to understand key events involved in the regeneration of periodontium. Polypeptide growth factors are a class of natural biological mediators which regulate key cellular events in tissue repair. Platelet-derived growth factor (PDGF) is the most thoroughly studied growth factor in periodontal regeneration. The present case series evaluate the effectiveness of platelet-derived growth factor (rh-PDGF-BB) in combination with beta-tricalcium phosphate (β-TCP) to achieve periodontal regeneration in 3 infrabony defects. [ABSTRACT FROM AUTHOR]

Therapies involving elevation of the endogenous suppressor cyclic AMP (cAMP) are currently used in the treatment of several chronic inflammatory disorders, including chronic obstructive pulmonary disease (COPD). Characteristics of COPD are airway obstruction, airway inflammation and airway remodelling, processes encompassed by increased airway smooth muscle mass, epithelial changes, goblet cell and submucosal gland hyperplasia. In addition to inflammatory cells, airway smooth muscle cells and (myo)fibroblasts, epithelial cells underpin a variety of key responses in the airways such as inflammatory cytokine release, airway remodelling, mucus hypersecretion and airway barrier function. Cigarette smoke, being next to environmental pollution the main cause of COPD, is believed to cause epithelial hyperpermeability by disrupting the barrier function. Here we will focus on the most recent progress on compartmentalized signalling by cAMP. In addition to G protein-coupled receptors, adenylyl cyclases, cAMP-specific phosphodiesterases (PDEs) maintain compartmentalized cAMP signalling. Intriguingly, spatially discrete cAMP-sensing signalling complexes seem also to involve distinct members of the A-kinase anchoring (AKAP) superfamily and IQ motif containing GTPase activating protein (IQGAPs). In this review, we will highlight the interaction between cAMP and the epithelial barrier to retain proper lung function and to alleviate COPD symptoms and focus on the possible molecular mechanisms involved in this process. Future studies should include the development of cAMP-sensing multiprotein complex specific disruptors and/or stabilizers to orchestrate cellular functions. Compartmentalized cAMP signalling regulates important cellular processes in the lung and may serve as a therapeutic target. [ABSTRACT FROM AUTHOR]

The native capacity of adult skeletal muscles to regenerate is vital to the recovery from physical injuries and dystrophic diseases. Currently, the development of therapeutic interventions has been hindered by the com-plex regulatory network underlying the process of muscle regeneration. Using a mouse model of skeletal mus-cle regeneration after injury, we identified hexamethylene bisacetamide inducible 1 (HEXIM1, also referred to as CLP-1), the inhibitory component of the positive transcription elongation factor b (P-TEFb) complex, as a pivotal regulator of skeletal muscle regeneration. Heximl-haplodeficient muscles exhibited greater mass and preserved function compared with those of WT muscles after injury, as a result of enhanced expansion of satellite cells. Transplanted Heximl -haplodeficient satellite cells expanded and improved muscle regeneration more effectively than WT satellite cells. Conversely, HEXIM1 overexpression restrained satellite cell prolifer-ation and impeded muscle regeneration. Mechanistically, dissociation of HEXIM1 from P-TEFb and subse-quent activation of P-TEFb are required for satellite cell proliferation and the prevention of early myogenic differentiation. These findings suggest a crucial role for the HEXIMl/P-TEFb pathway in the regulation of satellite cell-mediated muscle regeneration and identify HEXIM1 as a potential therapeutic target for degen-erative muscular diseases. [ABSTRACT FROM AUTHOR]

Mesothelium is the simple squamous epithelium covering all abdominal organs and the coeloms in which those organs reside. While the structural characteristics of this cell type were documented a century ago, its potential in development, disease, and wound healing is only now becoming apparent. In the embryo, mesothelia provide vasculogenic cells for the developing heart, lungs, and gut. Furthermore, adult mesothelial cells can be reactivated using thymosin β4 and mobilized to aid in tissue repair. Despite their positive role in development and repair, mesothelia are also susceptible to adhesion and tumor formation. With knowledge that the mesothelium is an important mediator of tissue repair as well as disease, it will be important to identify other factors like thymosin β4 that have the ability to potentiate these cells. Future use of chemical and genetic agents in conjunction with mesothelial cells will lead to enhanced therapeutic potential and mitigation of deleterious outcomes. [ABSTRACT FROM AUTHOR]

Subtotal cystectomy (STC; surgical removal of ~75% of the rat urinary bladder) elicits a robust proliferative response resulting in complete structural and functional bladder regeneration within 8-weeks. The goal of these studies was to characterize the early cellular response that mediates this regenerative phenomenon, which is unique among mammalian organ systems. STC was performed on eighteen 12-week-old female Fischer F344 rats. At 1, 3, 5 and 7-days post-STC, the bladder was harvested 2-hours after intraperitoneal injection of bromodeoxyuridine (BrdU). Fluorescent BrdU labeling was quantified in cells within the urothelium, lamina propria (LP), muscularis propria (MP) and serosa. Cell location was confirmed with fluorescently co-labeled cytokeratin, vimentin or smooth muscle actin (SMA), to identify urothelial, interstitial and smooth muscle cells, respectively. Expression of sonic hedgehog (Shh), Gli-1 and bone morphogenic factor-4 (BMP-4) were evaluated with immunochemistry. Three non-operated rats injected with BrdU served as controls. Less than 1% of cells in the bladder wall were labeled with BrdU in control bladders, but this percentage significantly increased by 5-8-fold at all time points post-STC. The spatiotemporal characteristics of the proliferative response were defined by a significantly higher percentage of BrdU-labeled cells within the urothelium at 1-day than in the MP and LP. A time-dependent shift at 3 and 5-days post-STC revealed significantly fewer BrdU-labeled cells in the MP than LP or urothelium. By 7-days the percentage of BrdU-labeled cells was similar among urothelium, LP and MP. STC also caused an increase in immunostaining for Shh, Gli-1 and BMP-4. In summary, the early stages of functional bladder regeneration are characterized by time-dependent changes in the location of the proliferating cell population, and expression of several evolutionarily conserved developmental signaling proteins. This report extends previous observations and further establishes the rodent bladder as an excellent model for studying novel aspects of mammalian organ regeneration. [ABSTRACT FROM AUTHOR]

The dystrophin-associated glycoprotein complex (DGC) is found at the muscle fiber sarcolemma and forms an essential structural link between the basal lamina and internal cytoskeleton. In a set of muscular dystrophies known as the dystroglycanopathies, hypoglycosylation of the DGC component α-dystroglycan results in reduced binding to basal lamina components, a loss in structural stability, and repeated cycles of muscle fiber degeneration and regeneration. The satellite cells are the key stem cells responsible for muscle repair and reside between the basal lamina and sarcolemma. In this study, we aimed to determine whether pathological changes associated with the dystroglycanopathies affect satellite cell function. In the Largemyd mouse dystroglycanopathy model, satellite cells are present in significantly greater numbers but display reduced proliferation on their native muscle fibers in vitro, compared with wild type. However, when removed from their fiber, proliferation in culture is restored to that of wild type. Immunohistochemical analysis of Largemyd muscle reveals alterations to the basal lamina and interstitium, including marked disorganization of laminin, upregulation of fibronectin and collagens. Proliferation and differentiation of wild-type satellite cells is impaired when cultured on substrates such as collagen and fibronectin, compared with laminins. When engrafted into irradiated tibialis anterior muscles of mdx-nude mice, wild-type satellite cells expanded on laminin contribute significantly more to muscle regeneration than those expanded on fibronectin. These results suggest that defects in α-dystroglycan glycosylation are associated with an alteration in the satellite cell niche, and that regenerative potential in the dystroglycanopathies may be perturbed. S TEM C ells 2012;30:2330-2341 [ABSTRACT FROM AUTHOR]

Cells sense a myriad of cues from their surrounding microenvironment to regulate their function. In recent years, it has become clear that physical and mechanical cues are as critical as biochemical factors in regulating cellular function. The geometry of the extracellular matrix (ECM), degree of cell spreading, and ECM rigidity all influence the physical connection between cells and their microenvironment and play a major role in regulating proliferation, differentiation, and migration. Leveraging these findings to promote specific cell behaviours will be paramount to realize the full potential of cellular therapies. In this review, I examine our current understanding of how mechanical cues—specifically, geometric control of cell shape and matrix rigidity—are transduced by stem cells to control their stemness, proliferation, and differentiation. The implications of these findings for vascular smooth muscle cell differentiation and cardiovascular tissue engineering will be highlighted. [ABSTRACT FROM PUBLISHER]

Abstract: To address the growing demand of small-diameter vascular grafts for cardiovascular disease, it is necessary to develop substitutes with bio-functionalities, such as anticoagulation, rapid endothelialization, and smooth muscle regeneration. In this study, the small-diameter tubular grafts (2.2 mm) were fabricated by electrospinning of biodegradable polymer polycaprolactone (PCL) followed by functional surface coating with an arginine-glycine-aspartic acid (RGD)-containing molecule. The healing characteristics of the grafts were evaluated by implanting them in rabbit carotid arteries for 2 and 4 weeks. Results showed that at both time points, all 10 of the RGD-modified PCL grafts (PCL-RGD) were patent, whereas 4 of the 10 non-modified PCL grafts were occluded due to thrombus formation. Scanning electron microscopy (SEM) data showed abundant platelets adhering on the surface of the midportion of the PCL grafts. In contrast, only few platelets were observed on the PCL-RGD surface, suggesting that RGD modification significantly improved the hemocompatibility of the PCL grafts. Histological analysis demonstrated enhanced cell infiltration and homogeneous distribution within the PCL-RGD grafts in comparison with the PCL grafts. Furthermore, immunofluorescence staining also showed a 3-fold increase of endothelial coverage of the PCL-RGD grafts than that of PCL grafts at those two time points. After 4-week implantation, 65.3 ± 7.6% of the surface area of the PCL-RGD grafts was covered by smooth muscle cell layer, which is almost 23% more than that on the PCL grafts. The present study indicates that RGD-modified PCL grafts exhibit an improved remodeling and integration capability in revascularization. [Copyright &y& Elsevier]

Background Fractional laser therapy ( FLT) has become a widely accepted modality for skin rejuvenation and has also been used in various other skin diseases. Objective To observe long-term histologic effects of nonablative and ablative FLT in the treatment of pigment disorders. Methods A randomized controlled observer-blinded study was performed in 18 patients with pigment disorders. Two similar test regions were randomized to receive FLT with intermittent topical bleaching or topical bleaching alone. Patients with ashy dermatosis ( AD) and postinflammatory hyperpigmentation ( PIH) were treated using nonablative 1,550-nm FLT (15 mJ/microbeam, 14-20% coverage), whereas patients with Becker's nevus ( BN) were treated with ablative 10,600-nm FLT (10 mJ/microbeam, 35-45% coverage) for three to five sessions. Biopsies were obtained 3 months after the last treatment. Results At follow-up, dermal fibrosis was observed in four of eight patients treated using ablative FLT and no patients treated using nonablative FLT ( p < .05). Conclusions Assuming that the dermal response is comparable in AD, PIH, and BN, at the given settings, ablative FLT may induce fibrosis, whereas treatment with nonablative FLT does not. Whether formation of fibrosis has to be regarded as dermal remodeling or a subtle subclinical form of scarring should be investigated in future research. [ABSTRACT FROM AUTHOR]

Abstract: Recent successes in regenerative medicine and tissue engineering of bladder and bladder-like neo-organs have leveraged regenerative constructs composed of a biodegradable scaffold seeded with a population of smooth muscle cells. We have shown that such smooth muscle cells are isolatable from adipose and other sources alternate to the primary organ. We hypothesize that this regenerative platform is not limited to regeneration of bladder and bladder-like neo-organs, but rather represents a foundational technology platform broadly applicable for regeneration of laminarly organized hollow organs. Using esophagus as an illustrative example in support of this hypothesis, we demonstrate that patch constructs composed of adipose-derived smooth muscle cells seeded on a biodegradable matrix catalyze complete regeneration of the esophageal wall in a rodent model of esophageal injury. By implication, such regenerative constructs may potentially be used to mediate the regeneration of any laminarly organized tubular organ. [Copyright &y& Elsevier]

Chronic hypoxia is an inciting factor for the development of pulmonary arterial hypertension. The mechanisms involved in the development of hypoxic pulmonary hypertension (HPH) include hypoxia-inducible factor 1 (HIF-1)-dependent transactivation of genes controlling pulmonary arterial smooth muscle cell (PASMC) intracellular calcium concentration ([Ca2+]i) and pH. Recently, digoxin was shown to inhibit HIF-1 transcriptional activity. In this study, we tested the hypothesis that digoxin could prevent and reverse the development of HPH. Mice were injected daily with saline or digoxin and exposed to room air or ambient hypoxia for 3 wk. Treatment with digoxin attenuated the development of right ventricle (RV) hypertrophy and prevented the pulmonary vascular remodeling and increases in PASMC [Ca2+]i, pH, and RV pressure that occur in mice exposed to chronic hypoxia. When started after pulmonary hypertension was established, digoxin attenuated the hypoxia-induced increases in RV pressure and PASMC pH and [Ca2+]i. These preclinical data support a role for HIF-1 inhibitors in the treatment of HPH. [ABSTRACT FROM AUTHOR]

Abstract: Insulin-like growth factor-1 (IGF-1) has been shown to induce potent mitogenic responses in various cell types, yet its sustained local delivery is still an underdeveloped domain in the clinic. We report here an engineered IGF-1 that facilitates extended local delivery to a site through its immobilization capacity within fibrin. Through recombinant fusion with a substrate sequence tag derived from α2-plasmin inhibitor (α2PI1-8), the resulting variant, α2PI1-8–IGF-1, was covalently incorporated into fibrin matrices during normal thrombin/factor XIIIa-mediated polymerization. Bioactivity of the variant was confirmed to be equivalent to wild type (WT) IGF-1 via IGF-1 receptor phosphorylation and cell proliferation studies in urinary tract-derived cells in 2-D. Assessment of functional retention within 3-D fibrin matrices demonstrated that incorporation of α2PI1–8–IGF-1 induced a 1.3- and 1.5-fold more robust proliferative response in smooth muscle cells (SMCs) than WT IGF-1 and negative control matrices, respectively, when release was not contained. Sustained α2PI1–8–IGF-1 availability at bladder lesion sites in vivo evoked a considerable increase in SMC proliferation and a favorable host tissue response after 28 days in rats. We conclude that the sustained local IGF-1 availability from fibrin provided by our variant protein enhances smooth muscle regeneration better than the WT form of the protein. [Copyright &y& Elsevier]

Abstract: Myocardial infarction (MI) is one of the leading causes of morbidity and mortality world-wide. Whether endogenous repair and regenerative ability could be augmented by drug administration is an important issue for generation of novel therapeutic approach. Recently it was reported that in mice pretreated with thymosin beta 4 (TB4) and subsequently subjected to experimental MI, a subset of epicardial cells differentiated into cardiomyocytes. In clinical settings, epicardial priming with TB4 prior to MI is impractical. Here we tested if TB4 treatment after MI could reprogram epicardium into cardiomyocytes and augment the epicardium''s injury response. Using epicardium genetic lineage trace line Wt1CreERT2/+ and double reporter line Rosa26mTmG/+, we found post-MI TB4 treatment significantly increased the thickness of epicardium and coronary capillary density. However, epicardium-derived cells did not adopt cardiomyocyte fate, nor did they migrate into myocardium to become coronary endothelial cells. Our result thus indicates that TB4 treatment after MI does not alter epicardial cell fate to include the cardiomyocyte lineage, providing both cautions and insights for the full exploration of the potential benefits of TB4 in the clinical settings. This article is part of a Special Issue entitled ‘Possible Editorial’. [Copyright &y& Elsevier]

Candidate stem/progenitor cells have been identified in mouse endometrium as label-retaining cells (LRCs). The role of endometrial stem/progenitor cells in initiating estrogen-stimulated endometrial growth in prepubertal and cycling mice was investigated following a single 17β-estradiol (E2) injection in bromodeoxyuridine (BrdU)-labeled and -chased (LRC), ovariectomised mice. Proliferating (BrdU+/Ki-67+) and mitotic (BrdU+/PH3+) epithelial LRCs were first detected in prepubertal mice 8 hours following E2 treatment, initiating the proliferative response. In contrast, all epithelial LRCs and 16% of epithelial cells in cycling mice proliferated within 2 hours. In cycling mice, 12% of stromal LRCs initiated a proliferative response 8 hours after E2. Proliferating epithelial LRCs and most stromal LRCs (85%) lacked estrogen receptor-α (ESR1). These findings suggest that endometrial epithelial LRCs function as stem/progenitor cells by receiving proliferative signals from neighboring ESR1+ niche cells to initiate the growth of the epithelium during development, while mature epithelial cells may undergo self-replication in cycling endometrium. [ABSTRACT FROM PUBLISHER]

Limited data exist on the molecular mechanisms that govern skeletal muscle regeneration in humans. This study characterized the early molecular alterations in humans to eccentric contractions (ECs), a stimulus known to induce a muscle regenerative response. Thirty-five subjects completed 100 ECs of the knee extensors with 1 leg, and muscle biopsies were taken from both legs 3 h post-EC. The sample from the non-EC leg served as the control. We first conducted a well-powered transcriptomic screen and network analysis. Our screen identified significant changes in several transcripts with functions relating to inflammation, cell growth, and proliferation. Network analysis then identified the transcription factor NF-κB as a key molecular element affected by ECs. A transcription factor ELISA, using nuclear extracts from EC and control muscle samples, showed a 1.6-fold increase in NF-κB DNA binding activity following ECs. Immunohistochemical experiments localized the majority of NF-κB-positive nuclei to cells in the interstitium, which stained positive for the pericyte markers NG2 proteoglycan and alkaline phosphatase. Our results provide the first evidence of NF-κB activation in human muscle following ECs and suggest a novel role for muscle residing pericytes in the early adaptive response to ECs. [ABSTRACT FROM AUTHOR]

Airway smooth muscle phenotype may be modulated in response to external stimuli under physiological and pathophysiological conditions. The effect of mechanical forces on airway smooth muscle phenotype were evaluated in vitro by suspending weights of 0.5 or 1 g from the ends of canine tracheal smooth muscle tissues, incubating the weighted tissues for 6 h, and then measuring the expression of the phenotypic marker protein, smooth muscle myosin heavy chain (SmMHC). Incubation of the tissues at a high load significantly increased expression of SmMHC compared with incubation at low load. Incubation of the tissues at a high load also decreased activation of PKB/Akt, as indicated by its phosphorylation at Ser 473. Inhibition of Akt or phosphatidylinositol-3,4,5 triphosphate-kinase increased SmMHC expression in tissues at low load but did not affect SmMHC expression at high load. IL-13 induced a significant increase in Akt activation and suppressed the expression of SmMHC protein at both low and high loads. The role of integrin signaling in mechanotransduction was evaluated by expressing a PINCH (LIM1-2) fragment in the muscle tissues that prevents the membrane localization of the integrin-binding IPP complex (ILK/PINCH/α-parvin), and also by expressing an inactive integrin-linked kinase mutant (ILK S343A) that inhibits endogenous ILK activity. Both mutants inhibited Akt activation and increased expression of SmMHC protein at low load but had no effect at high load. These results suggest that mechanical stress and IL-13 both act through an integrin-mediated signaling pathway to oppositely regulate the expression of phenotypic marker proteins in intact airway smooth muscle tissues. The stimulatory effects of mechanical stress on contractile protein expression oppose the suppression of contractile protein expression mediated by IL-13; thus the imposition of mechanical strain may inhibit changes in airway smooth muscle phenotype induced by inflammatory mediators. [ABSTRACT FROM AUTHOR]

The regenerative process of the perineurium and nerve function were examined using an in vivo model of perineurium resection in the rat sciatic nerve. Our hypothesis is that the regenerative process of the perineurium can be demonstrated by immunolabeling for tenascin-C and alpha smooth muscle actin after microsurgical resection of the perineurium in vivo. A total of 38 Lewis rats were used. Eight-week-old animals were assigned to one of two groups: the epi-perineurium removal group or the sham group. Under operative microscopy, the sciatic nerve was dissected from surrounding tissues at the thigh level from the ischial tuberosity to the fossa poplitea. The epi-perineurium was carefully removed by cutting circumferentially and stripping distally for 15 mm. For CatWalk® dynamic gait analysis, only right sciatic nerves underwent surgery; the left sciatic nerves were left intact. For pathological and electrophysiological tests, both the right and left sciatic nerves underwent surgery. Analysis of data was performed at each time interval with a two-group t-test. P < 0.05 was considered statistically significant. After resection of a 15-mm section of the epi-perineurium, immediate endoneurial swelling occurred in the outer portion and spread into the central portion. Although demyelination and axonal degeneration were found in the swollen area, remyelination and recovery of electrophysiological function were seen after regeneration of the perineurium. An immunohistological and electron microscopic study revealed that the perineurium regenerated via fusion of the residual interfascicular perineurium and endoneurial fibroblast-like cells of mesenchymal origin. CatWalk gait analysis showed not only motor paresis but also neuropathic pain during the early phases of this model. [ABSTRACT FROM AUTHOR]

Abstract: Tissue-engineered grafts for the urinary tract are being investigated for the potential treatment of several urologic diseases. These grafts, predominantly tubular-shaped, usually require in vitro culture prior to implantation to allow cell engraftment on initially cell-free scaffolds. We have developed a method to produce tubular-shaped collagen scaffolds based on plastic compression. Our approach produces a ready cell-seeded graft that does not need further in vitro culture prior to implantation. The tubular collagen scaffolds were in particular investigated for their structural, mechanical and biological properties. The resulting construct showed an especially high collagen density, and was characterized by favorable mechanical properties assessed by axial extension and radial dilation. Young modulus in particular was greater than non-compressed collagen tubes. Seeding densities affected proliferation rate of primary human bladder smooth muscle cells. An optimal seeding density of 106 cells per construct resulted in a 25-fold increase in Alamar blue-based fluorescence after 2 wk in culture. These high-density collagen gel tubes, ready seeded with smooth muscle cells could be further seeded with urothelial cells, drastically shortening the production time of graft for urinary tract regeneration. [Copyright &y& Elsevier]

Abstract: Fresh autologous tunica vaginalis was experimentally used for partial substitution of the excised urinary bladder wall in ten male mongrel dogs. The substituted areas of two dogs were examined macroscopically and histologically at 2, 4, 6, 8, and 10weeks after surgery. Two control dogs underwent partial cystectomy and primary wall closure without substitution. The regenerated transitional epithelium completely covered the substituted portion and smooth muscle regeneration was present at 6weeks. The bladder walls at the closure area of one control dog and at the substituted portions of two dogs at 10weeks were indistinguishable macroscopically from the native bladder with all layers of the bladder wall present histologically. According to the macroscopic and histological findings and simplicity of the technique, tunica vaginalis can be used as an alternative graft for bladder wall substitution. Calcification and bone metaplasia observed were similar to those found after using other tissue grafts. [ABSTRACT FROM AUTHOR]

Abstract: ATP-sensitive potassium (KATP) channel openers have been shown to protect against cellular damage in neurons, cardiac muscle, and kidney and to effectively reduce nonsteroidal anti-inflammatory drug (NSAID)-induced gastric damage in rats. We investigated the effects of KATP channel opener diazoxide on small intestinal injury induced in rats by indomethacin administration. The effect of glibenclamide, a KATP channel blocker, was also evaluated. Diazoxide (15, 45 and 135mg/kg) or glibenclamide (18mg/kg), were given by oral gavage 1h before and 6h after indomethacin treatment (20mg/kg p.o.). After 24h, macroscopic and histologic lesions, myeloperoxidase (MPO) activity and lipid peroxidation levels were evaluated. Diazoxide at 15mg/kg was ineffective, while at doses of 45mg/kg and 135mg/kg was able to significantly improve all damage parameters. Glibenclamide administration enhanced intestinal injury. These results show for the first time a beneficial effect of diazoxide in indomethacin-induced enteritis in the rat. Several mechanisms, such as oxidative phosphorylation uncoupling and hypermotility seem particularly important in NSAID-induced intestinal injury. Such events lead to increased mucosal permeability and to penetration of noxious lumen components, which ignite the inflammatory response. Since KATP channel openers were shown to protect against mitochondrial damage, to reduce intercellular permeability and to relax smooth muscle, we suggest that diazoxide could exert its beneficial effects by one or more of these actions. [ABSTRACT FROM AUTHOR]

Nippostrongylus brasiliensis infections generate pulmonary pathologies that can be associated with strong TH2 polarization of the host's immune response. We present data demonstrating N. brasiliensis-driven airway mucus production to be dependent on smooth muscle cell interleukin 4 receptor-α (IL-4Rα) responsiveness. At days 7 and 10 post infection (PI), significant airway mucus production was found in IL-4Rα−/lox control mice, whereas global knockout (IL-4Rα−/−) and smooth muscle-specific IL-4Rα-deficient mice (SM-MHCCre IL-4Rα−/lox) showed reduced airway mucus responses. Furthermore, interleukin (IL)-13 and IL-5 cytokine production in SM-MHCCre IL-4Rα−/lox mice was impaired along with a transient reduction in T-cell numbers in the lung. In vitro treatment of smooth muscle cells with secreted N. brasiliensis excretory-secretory antigen (NES) induced IL-6 production. Decreased protein kinase C (PKC)-dependent smooth muscle cell proliferation associated with cell cycle arrest was found in cells stimulated with NES. Together, these data demonstrate that both IL-4Rα and NES-driven responses by smooth muscle cells make important contributions in initiating TH2 responses against N. brasiliensis infections. [ABSTRACT FROM AUTHOR]

In tissue repair, fibroblasts migrate into the wound to produce and remodel extracellular matrix (ECM). Integrins are believed to be crucial for tissue repair, but their tissue-specific role in this process is poorly understood. Here, we show that mice containing a fibroblast-specific deletion of integrin β1 exhibit delayed cutaneous wound closure and less granulation tissue formation, including reduced production of new ECM and reduced expression of a-smooth muscle actin (α-SMA). Integrin-β1-deficient fibroblasts showed reduced expression of type I collagen and connective tissue growth factor, and failed to differentiate into myofibroblasts as a result of reduced α-SMA stress fiber formation. Loss of integrin β1 in adult fibroblasts reduced their ability to adhere to, to spread on and to contract ECM. Within stressed collagen matrices, integrin-β1-deficient fibroblasts showed reduced activation of latent TGFβ. Addition of active TGFβ alleviated the phenotype of integrin-β1-deficient mice. Thus integrin β1 is essential for normal wound healing, where it acts, at least in part, through a TGFβ-dependent mechanism in vivo. [ABSTRACT FROM AUTHOR]