Calpain- and talin-dependent control of microvascular pericyte contractility and cellular stiffness

Pericytes surround capillary endothelial cells and exert contractile forces modulating microvascular tone and endothelial growth. We previously described pericyte contractile phenotype to be Rho GTPase- and α-smooth muscle actin (αSMA)-dependent. However, mechanisms mediating adhesion-dependent shape changes and contractile force transduction remain largely equivocal. We now report that the neutral cysteine protease, calpain, modulates pericyte contractility and cellular stiffness via talin, an integrin-binding and F-actin associating protein. Digital imaging and quantitative analyses of living cells reveal significant perturbations in contractile force transduction detected via deformation of silicone substrata, as well as perturbations of mechanical stiffness in cellular contractile subdomains quantified via atomic force microscope (AFM)-enabled nanoindentation. Pericytes overexpressing GFP-tagged talin show significantly enhanced contractility (~ two-fold), which is mitigated when either the calpain-cleavage resistant mutant talin L432G or vinculin are expressed. Moreover, the cell-penetrating, calpain-specific inhibitor termed CALPASTAT reverses talin-enhanced, but not Rho GTP-dependent, contractility. Interestingly, our analysis revealed that CALPASTAT, but not its inactive mutant, alters contractile cell-driven substrata deformations while increasing mechanical stiffness of subcellular contractile regions of these pericytes. Altogether, our results reveal that calpain-dependent cleavage of talin modulates cell contractile dynamics, which in pericytes may prove instrumental in controlling normal capillary function or microvascular pathophysiology.National Science Foundation (U.S.) (CAREER Award)American Society for Engineering Education. National Defense Science and Engineering Graduate Fellowshi

Short-term efficacy of botulinum neurotoxin for spastic cerebral palsy: A prospective and controlled study with parental feedback

Introduction: Cerebral palsy (CP) is a group of disorders that affect muscle movement, tone and coordination. The reduction of spastic muscular paralysis can be obtained by intramuscular injection of botulinum neurotoxin type A (BTX-A).Objectives: This study aimed to evaluate the antispastic effect of BTX-A in children with spastic CP and to estimate the parents’ opinion about the effectiveness of BTX-A therapy.Material and methods: A group of 40 children was divided into the study (n = 24, BTX-A + rehabilitation) and the control group (n = 16, rehabilitation). The modified Ashworth scale (MAS) was used to assess the level of muscle tone. A survey method was used to determine the subjective opinion of the children’s parents regarding the effectiveness of BTX-A.Results: The BTX-A injections significantly reduced the level of muscle spasticity in children with CP (5.5 points in the study vs. 2.8 points in control; p = 0.008). The analysis from the univariate linear regression model showed children from the study group (B = 1.38, p = 0.005) and older children (B = –0.30, p = 0.046) influence the difference in obtained MAS scores. The best effect was obtained by combining the BTX-A injection with rehabilitation. Parents positively opinionated the use of BTX-A injections to improve functioning, decrease hypertonia, and facilitate carrying. 83% of parents noticed an improvement in their child’s functioning after the first injection of BTX-A and 92% would recommend BTX-A injections for CP.Conclusions: BTX-A injections lead to a reduction in spasticity in children with CP. The effects of therapy are particularly noticeable at the beginning of the treatment, and the most effective in the youngest patients. BTX-A injections combined with intensive rehabilitation contribute to an improved functional level for children with spastic CP

Pericyte actomyosin-mediated contraction at the cell-material interface can modulate the microvascular niche

Pericytes physically surround the capillary endothelium, contacting and communicating with associated vascular endothelial cells via cell–cell and cell–matrix contacts. Pericyte–endothelial cell interactions thus have the potential to modulate growth and function of the microvasculature. Here we employ the experimental finding that pericytes can buckle a freestanding, underlying membrane via actin-mediated contraction. Pericytes were cultured on deformable silicone substrata, and pericyte-generated wrinkles were imaged via both optical and atomic force microscopy (AFM). The local stiffness of subcellular domains both near and far from these wrinkles was investigated by using AFM-enabled nanoindentation to quantify effective elastic moduli. Substratum buckling contraction was quantified by the normalized change in length of initially flat regions of the substrata (corresponding to wrinkle contour lengths), and a model was used to relate local strain energies to pericyte contractile forces. The nature of pericyte-generated wrinkling and contractile protein-generated force transduction was further explored by the addition of pharmacological cytoskeletal inhibitors that affected contractile forces and the effective elastic moduli of pericyte domains. Actin-mediated forces are sufficient for pericytes to exert an average buckling contraction of 38% on the elastomeric substrata employed in these in vitro studies. Actomyosin-mediated contractile forces also act in vivo on the compliant environment of the microvasculature, including the basement membrane and other cells. Pericyte-generated substratum deformation can thus serve as a direct mechanical stimulus to adjacent vascular endothelial cells, and potentially alter the effective mechanical stiffness of nonlinear elastic extracellular matrices, to modulate pericyte–endothelial cell interactions that directly influence both physiologic and pathologic angiogenesis.National Science Foundation (U.S.) (CAREER Award)National Science Foundation (U.S.) (Chemical, Bioengineering, Environmental, and Transport Systems-0644846)National Institutes of Health (U.S.) (EY 19533)National Institutes of Health (U.S.) (EY 15125

Human NANOS1 Represses Apoptosis by Downregulating Pro-Apoptotic Genes in the Male Germ Cell Line

While two mouse NANOS paralogues, NANOS2 and NANOS3, are crucial for maintenance of germ cells by suppression of apoptosis, the mouse NANOS1 paralogue does not seem to regulate these processes. Previously, we described a human NANOS1 p.[(Pro34Thr);(Ser83del)] mutation associated with the absence of germ cells in seminiferous tubules of infertile patients, which might suggest an anti-apoptotic role of human NANOS1. In this study, we aimed to determine a potential influence of human NANOS1 on the maintenance of TCam-2 model germ cells by investigating proliferation, cell cycle, and apoptosis. Constructs encoding wild-type or mutated human NANOS1 were used for transfection of TCam-2 cells, in order to investigate the effect of NANOS1 on cell proliferation, which was studied using a colorimetric assay, as well as apoptosis and the cell cycle, which were measured by flow cytometry. RNA-Seq (RNA sequencing) analysis followed by RT-qPCR (reverse transcription and quantitative polymerase chain reaction) was conducted for identifying pro-apoptotic genes repressed by NANOS1. Here, we show that overexpression of NANOS1 downregulates apoptosis in TCam-2 cells. Moreover, we found that NANOS1 represses a set of pro-apoptotic genes at the mRNA level. We also found that the infertility-associated p.[(Pro34Thr);(Ser83del)] mutation causes NANOS1 to functionally switch from being anti-apoptotic to pro-apoptotic in the human male germ cell line. Thus, this report is the first to show an anti-apoptotic role of NANOS1 exerted by negative regulation of mRNAs of pro-apoptotic genes

Potential applications of using tissue-specific EVs in targeted therapy and vaccinology

Many cell types secrete spherical membrane bodies classified as extracellular vesicles (EVs). EVs participate in intercellular communication and are present in body fluids, including blood, lymph, and cerebrospinal fluid. The time of EVs survival in the body varies depending on the body’s localisation. Once the EVs reach cells, they trigger a cellular response. Three main modes of direct interaction of EVs with a target cell were described: receptor-ligand interaction mode, a direct fusion of EVs with the cellular membrane and EVs internalisation. Studies focused on the medical application of EVs. Medical application of EVs may require modification of their surface and interior. EVs surface was modified by affecting the parental cells or by the direct amendment of isolated EVs. The interior modification involved introducing materials into the cells or direct administrating isolated EVs. EVs carry proteins, lipids, fragments of DNA, mRNA, microRNA (miRNA) and long non-coding RNA. Because of EVs availability in liquid biopsy, they are potential diagnostic markers. Modified EVs could enhance the treatment of diseases such as colorectal cancer, Parkinson’s disease, leukaemia or liver fibrosis. EVs have specific tissue tropisms, which makes them convenient organ-directed carriers of nucleic acids, drugs and vaccines. In conclusion, recently published works have shown that EVs could become biomarkers and modern vehicles of advanced drug forms