Role of SARS-COV-2 and ACE2 in the pathophysiology of peripheral vascular diseases.

The occurrence of a novel coronavirus known as severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), created a serious challenge worldwide. SARS-CoV-2 has high infectivity, the ability to be transmitted even during the asymptomatic phase, and relatively low virulence, which has resulted in rapid transmission. SARS-CoV-2 can invade epithelial cells, hence, many patients infected with SARS-CoV-2 have suffered from vascular diseases (VDs) in addition to pulmonary manifestations. Accordingly, SARS-CoV-2 may can worsen the clinical condition of the patients with pre-existing VDs. Endothelial cells express angiotensin-converting enzyme 2 (ACE2). ACE2 is a biological enzyme that converts angiotensin (Ang)- 2 to Ang-(1-7). SARS-CoV-2 uses ACE2 as a cell receptor for viral entry. Thus, the SARS-CoV-2 virus promotes downregulation of ACE2, Ang-(1-7), and anti-inflammatory cytokines, as well as, an increase in Ang-2, resulting in pro-inflammatory cytokines. SARS-CoV-2 infection can cause hypertension, and endothelial damage, which can lead to intravascular thrombosis. In this review, we have concentrated on the effect of SARS-CoV-2 in peripheral vascular diseases (PVDs) and ACE2 as an enzyme in Renin-angiotensin aldosterone system (RAAS). A comprehensive search was performed on PubMed, Google Scholar, Scopus, using related keywords. Articles focusing on ("SARS-CoV-2", OR "COVID-19"), AND ("Vascular disease", OR "Peripheral vascular disease", OR interested disease name) with regard to MeSH terms, were selected. According to the studies, it is supposed that vascular diseases may increase susceptibility to severe SARS-CoV-2 infection due to increased thrombotic burden and endothelial dysfunction. Understanding SARS-CoV-2 infection mechanism and vascular system pathogenesis is crucial for effective management and treatment in pre-existing vascular diseases.

Peripheral Vascular Abnormalities in Anorexia Nervosa: A Psycho-Neuro-Immune-Metabolic Connection.

Immune, neuroendocrine, and autonomic nervous system dysregulation in anorexia nervosa lead to cardiovascular complications that can potentially result in increased morbidity and mortality. It is suggested that a complex non-invasive assessment of cardiovascular autonomic regulation-cardiac vagal control, sympathetic vascular activity, and cardiovascular reflex control-could represent a promising tool for early diagnosis, personalized therapy, and monitoring of therapeutic interventions in anorexia nervosa particularly at a vulnerable adolescent age. In this view, we recommend to consider in the diagnostic route, at least in the subset of patients with peripheral microvascular symptoms, a nailfold video-capillaroscopy as an easy not invasive tool for the early assessing of possible cardiovascular involvement.

Case Report: Novel SAVI-Causing Variants in STING1 Expand the Clinical Disease Spectrum and Suggest a Refined Model of STING Activation.

Gain-of-function mutations in STING1 cause the monogenic interferonopathy, SAVI, which presents with early-onset systemic inflammation, cold-induced vasculopathy and/or interstitial lung disease. We identified 5 patients (3 kindreds) with predominantly peripheral vascular disease who harbor 3 novel STING1 variants, p.H72N, p.F153V, and p.G158A. The latter two were predicted by a previous cryo-EM structure model to cause STING autoactivation. The p.H72N variant in exon 3, however, is the first SAVI-causing variant in the transmembrane linker region. Mutations of p.H72 into either charged residues or hydrophobic residues all led to dramatic loss of cGAMP response, while amino acid changes to residues with polar side chains were able to maintain the wild type status. Structural modeling of these novel mutations suggests a reconciled model of STING activation, which indicates that STING dimers can oligomerize in both open and closed states which would obliviate a high-energy 180° rotation of the ligand-binding head for STING activation, thus refining existing models of STING activation. Quantitative comparison showed that an overall lower autoactivating potential of the disease-causing mutations was associated with less severe lung disease, more severe peripheral vascular disease and the absence of a robust interferon signature in whole blood. Our findings are important in understanding genotype-phenotype correlation, designing targeted STING inhibitors and in dissecting differentially activated pathways downstream of different STING mutations.

Ubiquitous expression of Akt1 p.(E17K) results in vascular defects and embryonic lethality in mice.

Proteus syndrome is a progressive overgrowth disorder with vascular malformations caused by mosaic expression of the AKT1 c.49G > A, p.(E17K) activating variant which was predicted to cause lethality if expressed ubiquitously. To test that hypothesis, we used the ACTB-Cre gene to activate a conditional Akt1 p.(E17K) allele in the mouse. No offspring that was heterozygous for both Cre and the conditional allele (ßA-Akt1WT/flx) was viable. Fewer than expected numbers of ßA-Akt1WT/flx embryos were seen beginning at E11.5, but a few survived until E17.5. The phenotype ranged from mild to severe, but generally ßA-Akt1WT/flx embryos had fewer visible blood vessels and more hemorrhages than their wild-type littermates, which was suggestive of a vascular abnormality. Examination of E13.5 limb skin showed a primitive capillary network with increased branching complexity and abnormal patterning compared with wild-type skin. By E15.5, wild-type skin had undergone angiogenesis and formed a hierarchical network of remodeled vessels, whereas in ßA-Akt1WT/flx embryos, the capillary network failed to remodel. Mural cell coverage of the blood vessels was also reduced in ßA-Akt1WT/flx skin compared with that of wild type. Restricting expression of Akt1E17K to endothelial, cardiac or smooth muscle cells resulted in viable offspring and remodeled vasculature and did not recapitulate the ßA-Akt1WT/flx phenotype. We conclude that ubiquitous expression of Akt1E17K suppresses remodeling and inhibits the formation of a normal skin vasculature. We postulate that this failure prevents proper circulation necessary to support the growing embryo and that it is the result of interactions of multiple cell types with increased AKT signaling.

Dimethyl-2-oxoglutarate improves redox balance and mitochondrial function in muscle pericytes of individuals with diabetes mellitus.

AIMS/HYPOTHESIS: Treatment of vascular complications of diabetes remains inadequate. We reported that muscle pericytes (MPs) from limb muscles of vascular patients with diabetes mellitus display elevated levels of oxidative stress causing a dysfunctional phenotype. Here, we investigated whether treatment with dimethyl-2-oxoglutarate (DM-2OG), a tricarboxylic acid cycle metabolite with antioxidant properties, can restore a healthy metabolic and functional phenotype. METHODS: MPs were isolated from limb muscles of diabetes patients with vascular disease (D-MPs) and from non-diabetic control participants (ND-MPs). Metabolic status was assessed in untreated and DM-2OG-treated (1 mmol/l) cells using an extracellular flux analyser and anion-exchange chromatography-mass spectrometry (IC-MS/MS). Redox status was measured using commercial kits and IC-MS/MS, with antioxidant and metabolic enzyme expression assessed by quantitative RT-PCR and western blotting. Myogenic differentiation and proliferation and pericyte-endothelial interaction were assessed as functional readouts. RESULTS: D-MPs showed mitochondrial dysfunction, suppressed glycolytic activity and reduced reactive oxygen species-buffering capacity, but no suppression of antioxidant systems when compared with ND-MP controls. DM-2OG supplementation improved redox balance and mitochondrial function, without affecting glycolysis or antioxidant systems. Nonetheless, this was not enough for treated D-MPs to regain the level of proliferation and myogenic differentiation of ND-MPs. Interestingly, DM-2OG exerted a positive effect on pericyte-endothelial cell interaction in the co-culture angiogenesis assay, independent of the diabetic status. CONCLUSIONS/INTERPRETATION: These novel findings support the concept of using DM-2OG supplementation to improve pericyte redox balance and mitochondrial function, while concurrently allowing for enhanced pericyte-endothelial crosstalk. Such effects may help to prevent or slow down vasculopathy in skeletal muscles of people with diabetes. Graphical abstract.

The Association of Matrix Metalloproteinases with Chronic Kidney Disease and Peripheral Vascular Disease: A Light at the End of the Tunnel?

: Chronic Kidney Disease (CKD) represents a risk factor for fatal and nonfatal cardiovascular (CV) events, including peripheral vascular disease (PVD). This occurs because CKD encompasses several factors that lead to poor prognoses, mainly due to a reduction of the estimated glomerular filtration rate (eGFR), the presence of proteinuria, and the uremic inflammatory milieu. The matrix metalloproteinases (MMPs) are a group of zinc-containing endopeptidases implicated in extracellular matrix (ECM) remodeling, a systemic process in tissue homeostasis. MMPs play an important role in cell differentiation, angiogenesis, inflammation, and vascular damage. Our aim was to review the published evidence regarding the association between MMPs, PVD, and CKD to find possible common pathophysiological mechanisms. MMPs favor ECM deposition through the glomeruli, and start the shedding of cellular junctions and epithelial-mesenchymal transition in the renal tubules. MMP-2 and -9 have also been associated with the presence of systemic vascular damage, since they exert a pro-inflammatory and proatherosclerotic actions. An imbalance of MMPs was found in the context of PVD, where MMPs are predictors of poor prognoses in patients who underwent lower extremity revascularization. MMP circulating levels are increased in both conditions, i.e., that of CKD and PVD. A possible pathogenic link between these conditions is represented by the enhanced production of transforming growth factor-ß that worsens vascular calcifications and atherosclerosis and the development of proteinuria in patients with increased levels of MMPs. Proteinuria has been recognized as a marker of systemic vascular damage, and this may explain in part the increase in CV risk that is manifest in patients with CKD and PVD. In conclusion, MMPs can be considered a useful tool by which to stratify CV risk in patients with CKD and PVD. Further studies are needed to investigate the causal-relationships between MMPs, CKD, and PVD, and to optimize their prognostic and predictive (in response to treatments) roles.

Targeting Uremic Toxins to Prevent Peripheral Vascular Complications in Chronic Kidney Disease.

Chronic kidney disease (CKD) exhibits progressive kidney dysfunction and leads to disturbed homeostasis, including accumulation of uremic toxins, activated renin-angiotensin system, and increased oxidative stress and proinflammatory cytokines. Patients with CKD are prone to developing the peripheral vascular disease (PVD), leading to poorer outcomes than those without CKD. Cumulative evidence has showed that the synergy of uremic milieu and PVD could exaggerate vascular complications such as limb ischemia, amputation, stenosis, or thrombosis of a dialysis vascular access, and increase mortality risk. The role of uremic toxins in the pathogenesis of vascular dysfunction in CKD has been investigated. Moreover, growing evidence has shown the promising role of uremic toxins as a therapeutic target for PVD in CKD. This review focused on uremic toxins in the pathophysiology, in vitro and animal models, and current novel clinical approaches in reducing the uremic toxin to prevent peripheral vascular complications in CKD patients.

Applicability of Transcutaneous Oxygen Tension Measurement in the Assessment of Chronic Limb-Threatening Ischemia.

Transcutaneous oxygen tension measurement (TcPO2) is widely applied for the evaluation of chronic limb-threatening ischemia (CLTI). Nevertheless, studies that focused on the clinical value of TcPO2 have shown varying results. We identified factors that potentially play a role in TcPO2 measurement variation such as probe placement, probe temperature, and the use of a reference probe. In this review of the current literature, we assessed the application of these factors. A systematic search was conducted. Parameters that were assessed were probe placement, probe temperature, and mentioning and/or use of a reference probe. In total, 36 articles were eligible for analysis. In 24 (67%) studies, probes were placed on specific anatomical locations. Seven (19%) studies placed probes, regardless of the location of the ulcer, adjacent to an ischemic lesion or ulcer (perilesion). Selected temperature setting of the probe differed; in 18 (50%), a default probe temperature of 44°C was selected, and in 13 (36%), a different temperature was selected. In 31 (84%) studies, the use of a reference probe was not reported. Transcutaneous oxygen tension measurement is applied diversely in patients with CLTI. Homogeneity in TcPO2 protocols is warranted for reliable clinical application and to compare future TcPO2 research.

Histopathological and proteomic analyses identify integrin-ß1 as a potential mediator of phlebosclerosis in uremic patients.

BACKGROUND: Patients with uremia have an excessive mortality from cardiovascular disease (CVD). Arterial remodeling is mainly responsible for uremia-induced CVD and has been well studied, yet venous remodeling is poorly understood. Here we investigate the histopathology and proteomic profiles of venous remodeling in uremic patients. METHODS: Forearm cephalic veins were isolated from nine uremic patients during surgeries for arteriovenous fistula, and from nine healthy controls when applying surgical debridement. Hematoxylin-eosin, Masson's trichrome, von Kossa, and immunohistochemistry (IHC) against proliferating cell nuclear antigen were stained for histopathology. Isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis was executed to explore the proteome of the veins. The core regulatory protein was validated by western blot, IHC, and immunofluorescence. RESULTS: Phlebosclerosis, characterized by intimal rarefaction and medial thickening with disordered proliferation of vascular smooth muscle cells (VSMCs), was the prominent pathological manifestation of peripheral veins in uremic patients, while inflammatory cell infiltration, atherosclerosis or calcification were not obviously detected. iTRAQ analysis showed that 350 proteins were significantly changed in phlebosclerosis of uremic patients compared with healthy controls, of which integrin-ß1 (ITGß1) exhibited the strongest regulatory ability by intermolecular interaction network analysis. The enhanced ITGß1 expression was mainly co-expressed with the disordered proliferation of VSMCs while a little with vascular endothelial cells in the forearm cephalic veins of uremic patients. CONCLUSIONS: Phlebosclerosis is the prominent pathological manifestation in peripheral veins of uremic patients. This pathological alteration mainly attributes to the disordered proliferation of VSMCs, which is potentially mediated by ITGß1.

Aryl Hydrocarbon Receptor Nuclear Translocator in Vascular Smooth Muscle Cells Is Required for Optimal Peripheral Perfusion Recovery.

BACKGROUND: Limb ischemia resulting from peripheral vascular disease is a common cause of morbidity. Vessel occlusion limits blood flow, creating a hypoxic environment that damages distal tissue, requiring therapeutic revascularization. Hypoxia-inducible factors (HIFs) are key transcriptional regulators of hypoxic vascular responses, including angiogenesis and arteriogenesis. Despite vascular smooth muscle cells' (VSMCs') importance in vessel integrity, little is known about their functional responses to hypoxia in peripheral vascular disease. This study investigated the role of VSMC HIF in mediating peripheral ischemic responses. METHODS AND RESULTS: We used ArntSMKO mice with smooth muscle-specific deletion of aryl hydrocarbon receptor nuclear translocator (ARNT, HIF-1ß), required for HIF transcriptional activity, in a femoral artery ligation model of peripheral vascular disease. ArntSMKO mice exhibit impaired perfusion recovery despite normal collateral vessel dilation and angiogenic capillary responses. Decreased blood flow manifests in extensive tissue damage and hypoxia in ligated limbs of ArntSMKO mice. Furthermore, loss of aryl hydrocarbon receptor nuclear translocator changes the proliferation, migration, and transcriptional profile of cultured VSMCs. ArntSMKO mice display disrupted VSMC morphologic features and wrapping around arterioles and increased vascular permeability linked to decreased local blood flow. CONCLUSIONS: Our data demonstrate that traditional vascular remodeling responses are insufficient to provide robust peripheral tissue reperfusion in ArntSMKO mice. In all, this study highlights HIF responses to hypoxia in arteriole VSMCs critical for the phenotypic and functional stability of vessels that aid in the recovery of blood flow in ischemic peripheral tissues.

[Biological aspects and clinical application of bone-marrow stem cells in critical lower limb ischaemia]. / Biologicheskie aspekty i klinicheskoe primenenie stvolovykh kletok pri kriticheskoi ishemii nizhnikh konechnostei.

Critical lower-limb ischaemia (CLLI) is associated with high risk of limb loss and a lethal outcome, as well as with decreased quality of life. The underlying cause of the disease is imbalance between blood supply of ischaemized tissues and their metabolic demands. Restoration of adequate perfusion with the help of standard medicamentous therapy or operative treatment is often inefficient or impossible. Cell therapy (CT) is a novel strategy making it possible to stimulate the growth of the microvascular bed and the mechanisms of molecular-cellular repair. One of the most promising trends is the use of bone marrow stem cells (BMSCs). Sufficient evidence concerning safety and relative efficacy of CT has by now been accumulated. The existing differences in the results of studies are related to numerous peculiarities of both CT itself and methods of its application. The present review is dedicated to contemporary notions of the biology of BMSCs, assessment of safety of CT and the results of its application in treatment of CLLI.

Editor's Highlight: PCB126 Exposure Increases Risk for Peripheral Vascular Diseases in a Liver Injury Mouse Model.

The liver is vital for xenobiotic and endobiotic metabolism. Previously, we demonstrated that a compromised liver worsened toxicity associated with exposure to polychlorinated biphenyls (PCBs), through disruption of energy homeostasis. However, the role of a compromised liver in defining dioxin-like PCB126 toxicity on the peripheral vasculature and associated inflammatory diseases is yet to be studied. This study investigated the effects of PCB126 on vascular inflammation linked to hepatic dysfunction utilizing a liver injury mouse model. Male C57Bl/6 mice were fed either an amino acid control diet (CD) or a methionine-choline deficient (MCD) diet in this 14-week study. Mice were exposed to PCB126 (0.5 mg/kg) and analyzed for inflammatory, calorimetric and metabolic parameters. MCD diet-fed mice demonstrated steatosis, indicative of a compromised liver. Mice fed the MCD-diet and subsequently exposed to PCB126 manifested lower body fat mass, increased liver to body weight ratio and alterations in hepatic gene expression related to lipid and carbohydrate metabolism, implicating metabolic disturbances. PCB126-induced steatosis irrespective of the diet type, but only the MCD + PCB126 group exhibited steatohepatitis and fibrosis. Furthermore, PCB126 exposure in MCD-fed mice led to increased plasma inflammatory markers such as Icam-1, plasminogen activator inhibitor-1 and proatherogenic trimethylamine-N-oxide, suggesting inflammation of the peripheral vasculature that is characteristic of atherosclerosis. Taken together, our data provide new evidence of a link between a compromised liver, PCB-mediated hepatic inflammation and vascular inflammatory markers, suggesting that environmental pollutants can promote crosstalk between different organ systems, leading to inflammatory disease pathologies.

Overexpression of Thioredoxin1 enhances functional recovery in a mouse model of hind limb ischemia.

BACKGROUND: There is keen interest in finding nonsurgical treatments for peripheral vascular disease (PVD). Previously, we demonstrated that selective activation of Thioredoxin1 (Trx1), a 12-kDa cytosolic protein, initiates redox-dependent signaling and promotes neovascularization after ischemic heart disease. Therefore, Trx1 might possess immense potential to not only treat murine hind limb ischemia (HLI) through effective angiogenesis but also provide PVD patients with nonsurgical therapy to enhance neovascularization and improve blood perfusion. METHODS: To determine whether activation of Trx1 increases blood perfusion in HLI, two different strategies were used-gene therapy and transgenic model system. In adenoviral-mediated gene therapy, 8- to 12-wk-old mice were divided into two groups: (1) control Adeno-LacZ (Ad-LacZ) and (2) Adeno-Thiroedoxin1 (Ad-Trx1). The mice underwent surgical intervention to induce right HLI followed by injection with Ad-LacZ or Ad-Trx1, respectively. In the second strategy, we used wild-type and transgenic mice overexpressing Trx1 (Trx1Tg/+). All the animals underwent Doppler imaging for the assessment of limb perfusion followed by immunohistochemistry and Western blot analysis. RESULTS: Significant increases in perfusion ratio were observed in all the Trx1 overexpressed groups compared with their corresponding controls. Expressions of heme oxygenase-1, vascular endothelial growth factor, and the vascular endothelial growth factor receptors Flk-1 and Flt-1 were increased in Trx1 overexpressed mice compared with their respective controls. Blood perfusion in the ischemic limb gradually improved and significantly recovered in Trx1Tg/+ and Ad-Trx1 groups compared with their corresponding controls. The capillary and arteriolar density in the ischemic zone were found to be higher in Trx1Tg/+ group compared with wild type. CONCLUSIONS: The overall outcomes of our study demonstrate that Trx1 enhances blood perfusion and increases angiogenic protein expression in a rodent HLI model. These results suggest that Trx1 is a potential target for clinical trials and drug therapy for the treatment of PVD.

An update on biomarker discovery and use in systemic sclerosis.

INTRODUCTION: Systemic sclerosis (SSc) is an autoimmune disease characterized by excessive extracellular matrix deposition in the skin and internal organs. Three major abnormalities, autoimmunity, vasculopathy, and fibrosis, are considered to play important roles in the pathophysiology of SSc. SSc is a heterogeneous disease with clinical features, disease progress, therapeutic response, and prognosis. Therefore, identification of biomarkers, which can predict the course of the disease, is required for the progress of clinical practice. Areas covered: This review focuses on various SSc biomarkers for diagnosis, disease severity, and activity, as well as newly emerging and promising SSc biomarkers. Search was through PubMed and we focus on the papers in the last 5 years. Expert commentary: Biomarkers for SSc can be categorized into several groups: activity biomarker, severity biomarker, predictive biomarker, and biomarkers for specific clinical features (skin fibrosis, lung fibrosis, pulmonary arterial hypertension, peripheral vasculopathy, gastrointestinal, and malignancy). Some chemokines, such as CCL2 and CXCL4, have been identified as biomarkers of skin and lung fibrosis in SSc. In addition, anti-RNA polymerase III antibody has been noted to be a predictive biomarker of gastric antral vascular ectasia and malignancy.

[Diabetic foot syndrome: importance of calf muscles MR spectroscopy in the assessment of limb ischemia and effect of revascularization]. / Syndrom diabetické nohy: význam MR spektroskopie lýtkových svalu pro hodnocení koncetinové ischemie a efektu revaskularizace.

AIM: The standard method for assessment of effect of revascularization in patients with diabetic foot (DF) and critical limb ischemia (CLI) is transcutaneous oxygen pressure (TcPO2). Phosphorus magnetic resonance spectroscopy (31P MRS) enables to evaluate oxidative muscle metabolism that could be impaired in patients with diabetes and its complications. The aim of our study was to compare MRS of calf muscle between patients with DF and CLI and healthy controls and to evaluate the contribution of MRS in the assessment of the effect of revascularization. METHODS: Thirty-four diabetic patients with DF and CLI treated either by autologous cell therapy (ACT; 15 patients) or percutaneous transluminal angioplasty (PTA; 12 patients) in our foot clinic during 2013-2016 and 19 healthy controls were included into the study. TcPO2 measurement was used as a standard method of non-invasive evaluation of limb ischemia. MRS examinations were performed using the whole-body 3T MR system 1 day before and 3 months after the procedure. Subjects were examined in a supine position with the coil fixed under the m. gastrocnemius. MRS parameters were obtained at rest and during the exercise period. Rest MRS parameters of oxidative muscle metabolism such as phosphocreatine (PCr), inorganic phosphate (Pi), phosphodiesters (PDE), adenosine triphosphate (ATP), dynamic MRS parameters such as recovery constant PCr (τPCr) and mitochondrial capacity (Qmax), and pH were compared between patients and healthy controls, and also before and 3 months after revascularization. RESULTS: Patients with CLI had significantly lower PCr/Pi (p < 0.001), significantly higher Pi and pH (both p < 0.01), significantly lower Qmax and prolonged τPCr (both p < 0.001) in comparison with healthy controls. We observed a significant improvement in TcPO2 at 3 months after revascularization (from 26.4 ± 11.7 to 39.7 ± 17.7 mm Hg, p < 0.005). However, the rest MRS parameters did not change significantly after revascularization. In individual cases we observed improvement of dynamic MRS parameters. There was no correlation between MRS parameters and TcPO2 values. CONCLUSION: Results of our study show impaired oxidative metabolism of calf muscles in patients with CLI in comparison with healthy controls. We observed an improvement in dynamic MRS parameters in individual cases; this finding should be verified in a large number of patients during longer follow-up.Key words: autologous cell therapy - critical limb ischemia - diabetic foot - MR spectroscopy.

The RNA-binding protein caper is required for sensory neuron development in Drosophila melanogaster.

BACKGROUND: Alternative splicing mediated by RNA-binding proteins (RBPs) is emerging as a fundamental mechanism for the regulation of gene expression. Alternative splicing has been shown to be a widespread phenomenon that facilitates the diversification of gene products in a tissue-specific manner. Although defects in alternative splicing are rooted in many neurological disorders, only a small fraction of splicing factors have been investigated in detail. RESULTS: We find that the splicing factor Caper is required for the development of multiple different mechanosensory neuron subtypes at multiple life stages in Drosophila melanogaster. Disruption of Caper function causes defects in dendrite morphogenesis of larval dendrite arborization neurons and neuronal positioning of embryonic proprioceptors, as well as the development and maintenance of adult mechanosensory bristles. Additionally, we find that Caper dysfunction results in aberrant locomotor behavior in adult flies. Transcriptome-wide analyses further support a role for Caper in alternative isoform regulation of genes that function in neurogenesis. CONCLUSIONS: Our results provide the first evidence for a fundamental and broad requirement for the highly conserved splicing factor Caper in the development and maintenance of the nervous system and provide a framework for future studies on the detailed mechanism of Caper-mediated RNA regulation. Developmental Dynamics 246:610-624, 2017. © 2017 Wiley Periodicals, Inc.

Use of p53-Silenced Endothelial Progenitor Cells to Treat Ischemia in Diabetic Peripheral Vascular Disease.

BACKGROUND: Peripheral vascular disease is a major diabetes mellitus-related complication. In this study, we noted that expressions of proapoptotic p53 gene and its downstream cascade gene such as p21 are upregulated in hyperglycemia. Therefore, we investigated whether p53- and p21-silenced endothelial progenitor cells (EPCs) were able to survive in hyperglycemic milieu, and whether transplantation of either p53 knockout (KO) or p21KO or p53- and p21-silenced EPCs could improve collateral vessel formation and blood flow in diabetic vaso-occlusive peripheral vascular disease mouse models. METHODS AND RESULTS: We transplanted p53 and p21KO mouse EPCs (mEPCs) into streptozotocin-induced diabetic (type 1 diabetes mellitus model) C57BL/6J and db/db (B6.BKS(D)-Leprdb/J) (type 2 model) post-femoral artery occlusion. Similarly, Ad-p53-silenced and Ad-p21-silenced human EPCs (CD34+) cells were transplanted into streptozotocin-induced diabetic NOD.CB17-Prkdcscid/J mice. We measured blood flow at 3, 7, and 10 days and hindlimb muscles were obtained postsacrifice for mRNA estimation and CD31 staining. Enhanced blood flow was noted with delivery of p53 and p21KO mEPCs in streptozotocin-induced diabetic C57BL/6J mice. Similar results were obtained when human Ad-p53shEPCs(CD34+) and Ad-p21shEPCs(CD34+) were transplanted into streptozotocin-induced nonobese diabetic severe combined immunodeficiency mice. Gene expression analysis of p53 and p21KO EPCs transplanted hindlimb muscles showed increased expression of endothelial markers such as endothelial nitric oxide synthase, vascular endothelial growth factor A, and platelet endothelial cell adhesion molecule 1. Similarly, quantitative reverse transcriptase polymerase chain reaction of human Ad-p53shEPCs (CD34+)- and Ad-p21shEPCs (CD34+)-transplanted hindlimb muscles also showed increased expression of endothelial markers such as vascular endothelial growth factor A, noted primarily in the p53-silenced EPCs group. However, such beneficial effect was not noted in the db/db type 2 diabetic mouse models. CONCLUSIONS: Transient silencing of p53 using adenoviral vector in EPCs may have a therapeutic role in diabetic peripheral vascular disease.

Assessment of microvascular endothelial function in type 1 diabetes using laser speckle contrast imaging.

OBJECTIVE: To test whether laser speckle contrast imaging (LSCI) coupled with physiological post-occlusive reactive hyperemia (PORH) and pharmacological iontophoresis of acetylcholine (ACh) as local vasodilator stimuli could distinguish between cutaneous microvascular responses of Type 1 Diabetes (T1DM)'s patients with endothelial dysfunction and that of healthy controls. METHODS: Patients with T1DM aged ≥12years completed a clinical-epidemiological questionnaire. Data detailing patients' such as daily insulin dose, duration of diabetes, and use of pharmaceuticals such as antihypertensive drugs and statins that could interfere with endothelial function were obtained. Vascular reactivity was assessed in the forearm by LSCI and PORH at baseline and during iontophoresis of ACh using increasing anodic currents of 30, 60, 90, 120, 150 and 180µA in 10second intervals. RESULTS: This study included 50 patients with T1DM and 30 control subjects. The mean resting flux did not differ between patients and control subjects. T1DM patients exhibited endothelial dysfunction upon challenge with physiological or pharmacological stimuli. The microvascular response to both ACh and PORH (i.e., maximum response at peak and amplitude) were significantly reduced in patients with diabetes compared with control subjects (p<0.001). CONCLUSION: We demonstrated that endothelium-dependent skin microvascular vasodilator responses are significantly impaired in patients with T1DM compared to healthy subjects investigated using LSCI coupled with ACh iontophoresis and PORH. Additionally, we find that LSCI is a promising methodology for studying physiological vascular reactivity in T1DM.

Diabetic Microvascular Disease and Pulmonary Fibrosis: The Contribution of Platelets and Systemic Inflammation.

Diabetes is strongly associated with systemic inflammation and oxidative stress, but its effect on pulmonary vascular disease and lung function has often been disregarded. Several studies identified restrictive lung disease and fibrotic changes in diabetic patients and in animal models of diabetes. While microvascular dysfunction is a well-known complication of diabetes, the mechanisms leading to diabetes-induced lung injury have largely been disregarded. We described the potential involvement of diabetes-induced platelet-endothelial interactions in perpetuating vascular inflammation and oxidative injury leading to fibrotic changes in the lung. Changes in nitric oxide synthase (NOS) activation and decreased NO bioavailability in the diabetic lung increase platelet activation and vascular injury and may account for platelet hyperreactivity reported in diabetic patients. Additionally, the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway has been reported to mediate pancreatic islet damage, and is implicated in the onset of diabetes, inflammation and vascular injury. Many growth factors and diabetes-induced agonists act via the JAK/STAT pathway. Other studies reported the contribution of the JAK/STAT pathway to the regulation of the pulmonary fibrotic process but the role of this pathway in the development of diabetic lung fibrosis has not been considered. These observations may open new therapeutic perspectives for modulating multiple pathways to mitigate diabetes onset or its pulmonary consequences.

Mitochondrial Respiration after One Session of Calf Raise Exercise in Patients with Peripheral Vascular Disease and Healthy Older Adults.

PURPOSE: Mitochondria are essential for energy production in the muscle cell and for this they are dependent upon a sufficient supply of oxygen by the circulation. Exercise training has shown to be a potent stimulus for physiological adaptations and mitochondria play a central role. Whether changes in mitochondrial respiration are seen after exercise in patients with a reduced circulation is unknown. The aim of the study was to evaluate the time course and whether one session of calf raise exercise stimulates mitochondrial respiration in the calf muscle of patients with peripheral vascular disease. METHODS: One group of patients with peripheral vascular disease (n = 11) and one group of healthy older adults (n = 11) were included. Patients performed one session of continuous calf raises followed by 5 extra repetitions after initiation of pain. Healthy older adults performed 100 continuous calf raises. Gastrocnemius muscle biopsies were collected at baseline and 15 minutes, one hour, three hours and 24 hours after one session of calf raise exercise. A multi substrate (octanoylcarnitine, malate, adp, glutamate, succinate, FCCP, rotenone) approach was used to analyze mitochondrial respiration in permeabilized fibers. Mixed-linear model for repeated measures was used for statistical analyses. RESULTS: Patients with peripheral vascular disease have a lower baseline respiration supported by complex I and they increase respiration supported by complex II at one hour post-exercise. Healthy older adults increase respiration supported by electron transfer flavoprotein and complex I at one hour and 24 hours post-exercise. CONCLUSION: Our results indicate a shift towards mitochondrial respiration supported by complex II as being a pathophysiological component of peripheral vascular disease. Furthermore exercise stimulates mitochondrial respiration already after one session of calf raise exercise in patients with peripheral vascular disease and healthy older adults. TRIAL REGISTRATION: ClinicalTrials.gov NCT01842412.