Endothelial Wound Healing Research Articles

The Role of Mesenchymal Stem Cells for Corneal Endothelial Regeneration: A Systematic Review.

A single layer of tightly spaced cells, known as the endothelium, rests on the posterior side of the cornea. This endothelium regulates the stroma's relative dehydration, which is essential for corneal clarity. Cell therapy is an innovative method being used to repair various corneal abnormalities. Mesenchymal stem cells (MSCs) are now one of the most significant types of stem cells scientists have studied. This study aimed to evaluate the role of MSCs for corneal endothelial regeneration. A systematic review was performed by searching for articles from reputable databases with many study-type references, including PubMed, Cochrane Library, Science Direct, and Google Scholar, up to January 2024. The resulting data were displayed using the 2020 PRISMA flowchart and evaluated using the PRISMA 2020 checklist. Most of the included studies were in vivo and used topical application and anterior chamber injection as the administration routes. Based on the findings of this review, MSCs increased corneal endothelial cell density, improved the defect area and corneal transparency, facilitated endothelial cell regeneration and wound healing, and decreased neovascularization and corneal pro-inflammatory cytokines as compared to controls. Administration of MSCs into the anterior chamber could increase regeneration and proliferation of corneal endothelial tissue.

The role of MDM2 in angiogenesis: implications for endothelial tip cell formation.

In the present study, we examined the role of MDM2 in the angiogenesis process and its potential association with the sprouting of endothelial tip cells. To address this, we performed hypoxia-treated gastric cancer cells (HGC-27) to quantitative RT-PCR and Western blot analysis to measure the levels of MDM2 and VEGF-A mRNA and protein expression. Subsequently, we employed siRNA to disrupt MDM2 expression, followed by hypoxia treatment. The expression levels of MDM2 and VEGF-A mRNA and protein were subsequently reassessed. Additionally, ELISA was utilized to quantify the secretion levels of VEGF-A in each experimental group. A conditioned medium derived from HGC-27 cells treated with different agents was employed to assess its influence on the formation of EA.hy926 endothelial tip cells, using various techniques including Transwell plates migration assays, wound healing experiments, vascular formation assays, scanning electron microscopy, and immunofluorescence staining. These findings demonstrated that the in vitro knockdown of MDM2 in the conditioned medium exhibited significant inhibitory effects on endothelial cell migration, wound healing, and vascular formation. Additionally, the intervention led to a reduction in the presence of CD34+ tip cells and the formation of filopodia in endothelial cells, while partially restoring the integrity of tight junctions. Subsequent examination utilizing RNA-seq revealed that the suppression of MDM2 in HGC-27 cells resulted in the downregulation of the PI3K/AKT signaling pathway. Consequently, this downregulation led to an elevation in angiogenic effects induced by hypoxia.

Abstract Tu034: Endothelial cell regeneration by secretome obtained from P53 silenced bone marrow derived mesenchymal stromal cells (BM-MSCs)

Introduction: Diabetes is associated with impaired endothelial function. Studies have demonstrated that endothelial progenitor cells (EPCs) and mature endothelial cells (EC) are susceptible to apoptosis in hyperglycemic environment. Hyperglycemia leads to p53 gene over-expression and p53 gene silencing prior to hyperglycemia exposure prevents cellular senescence, improves mitochondrial function and helps vascular regeneration.. However, it is not possible to obtain sufficient quantities of secretome for therapeutic purposes from hematopoietic CD34+ve stem cells (HSCs). Our goal in this project was to test whether conditioned media (CM) obtained from p53 silenced human stem cells (other than CD34+ve cells) can also improve endothelial regeneration exposed to hyperglycemia. We compared CM obtained from adipose and bone marrow derived mesenchymal stem cells on endothelial regeneration Methods: Ad-human-P53 (TP53)-shRNA was used to silence P53 and Ad-scrambled-null-shRNA was used as control in bone marrow derived mesenchymal stromal cells. To collect conditioned media BM-MSCs were cultured in exosome free FBS media for 5 days, following transduction with Ad-p53sh or Ad-null, as the case may be. After transduction, cells were grown in media containing 2% exosome free FBS for only 48h and the collected supernatant was concentrated 10-fold to obtain the CM. Endothelial regeneration was tested using endothelial wound healing assay kit ( from Cell Biolabs # CBA-120-T). We compared CM obtained from null and p53sh in adipose and bone marrow derived MSCs on human endothelial cells in normal and high glucose conditions Results: We observed that CM from bone marrow derived MSCs increased the proliferation of endothelial cells in both normal and high glucose conditions, more so than adipose derived MSCs. We are currently studying the role of exosomes from this CM on cell proliferation and we are also analyzing the secretome (exosomal and non- exosomal fraction) by mass spectroscopy. Conclusion: P53 silenced BMMSC-CM appears to improve endothelial regeneration in hyperglycemia similar to the effect of p53-silenced HSCs. This maybe secondary to the fact that both these cells are derived from bone marrow and p53 silencing improves mitochondrial function. The CM obtained from BM-MSCs are in sufficient quantities and may have a prominent therapeutic role in treating diabetes vascular complications such as diabetic wound healing.

Transcription factor 4 promotes increased corneal endothelial cellular migration by altering microtubules in Fuchs endothelial corneal dystrophy

Fuchs endothelial corneal dystrophy (FECD) is a complex corneal disease characterized by the progressive decline and morphological changes of corneal endothelial cells (CECs) that leads to corneal edema and vision loss. The most common mutation in FECD is an intronic CTG repeat expansion in transcription factor 4 (TCF4) that leads to its altered expression. Corneal endothelial wound healing occurs primarily through cell enlargement and migration, and FECD CECs have been shown to display increased migration speeds. In this study, we aim to determine whether TCF4 can promote cellular migration in FECD CECs. We generated stable CEC lines derived from FECD patients that overexpressed different TCF4 isoforms and investigated epithelial-to-mesenchymal (EMT) expression, morphological analysis and cellular migration speeds. We found that full length TCF4-B isoform overexpression promotes cellular migration in FECD CECs in an EMT-independent manner. RNA-sequencing identified several pathways including the negative regulation of microtubules, with TUBB4A (tubulin beta 4A class IVa) as the top upregulated gene. TUBB4A expression was increased in FECD ex vivo specimens, and there was altered expression of cytoskeleton proteins, tubulin and actin, compared to normal healthy donor ex vivo specimens. Additionally, there was increased acetylation and detyrosination of microtubules in FECD supporting that microtubule stability is altered in FECD and could promote cellular migration. Future studies could be aimed at investigating if targeting the cytoskeleton and microtubules would have therapeutic potential for FECD by promoting cellular migration and regeneration.

Abstract 2058: Endothelial Cell Regeneration By Secretome Obtained From P53 Silenced Bone Marrow Derived MSCs (BMSCs).

Introduction: Diabetes is associated with impaired endothelial function. Studies have demonstrated that endothelial progenitor cells (EPCs) and mature endothelial cells (EC) are susceptible to apoptosis in hyperglycemic environment. Hyperglycemia promotes p53 gene over-expression and p53 gene silencing prior to hyperglycemia exposure prevents cellular senescence and helps vascular regeneration. Previously we have shown that p53 silenced human CD34+ve progenitor cell therapy and even associated cell derived secretome, helped tissue regeneration. Our goal in this project was to test whether conditioned media (CM) obtained from p53 silenced human stem cells (other than CD34+ve cells) can also improve endothelial regeneration exposed to hyperglycemia. We compared CM obtained from adipose and bone marrow derived mesenchymal stem cells on endothelial regeneration Methods: Ad-human-P53 (TP53)-shRNA was used to silence P53 and Ad-scrambled-null-shRNA was used as control in bone marrow derived mesenchymal stromal cells. To collect conditioned media BM-MSCs were cultured in exosome free FBS media for 5 days, following transduction with Ad-p53sh or Ad-null, as the case may be. After transduction, cells were grown in media containing 2% exosome free FBS for only 48h and the collected supernatant was concentrated 10-fold to obtain the CM. Endothelial regeneration was tested using endothelial wound healing assay kit ( from Cell Biolabs # CBA-120-T). We compared CM obtained from null and p53sh in adipose and bone marrow derived mesenchymal stem cells on human endothelial cells in normal and high glucose conditions Results: Proliferation rate of endothelial cells was enhanced when we used CM from bone marrow derived cells as compared to CM from adipose derived mesenchymal stem cells, by nearly two fold. We are currently analyzing the secretome by mass spectroscopy. Conclusion: P53 silencing appears to improve endothelial regeneration on using CM obtained from bone marrow derived stem cells. This maybe secondary to improved mitochondrial function post p53 gene silencing. The CM thus obtained are in sufficient quantities and may have therapeutic role in treating diabetes vascular complications such as diabetic wound healing.

Complement MASP-1 Modifies Endothelial Wound Healing.

Endothelial wound-healing processes are fundamental for the maintenance and restoration of the circulatory system and are greatly affected by the factors present in the blood. We have previously shown that the complement protein mannan-binding lectin-associated serine protease-1 (MASP-1) induces the proinflammatory activation of endothelial cells and is able to cooperate with other proinflammatory activators. Our aim was to investigate the combined effect of mechanical wounding and MASP-1 on endothelial cells. Transcriptomic analysis showed that MASP-1 alters the expression of wound-healing-related and angiogenesis-related genes. Both wounding and MASP-1 induced Ca2+ mobilization when applied individually. However, MASP-1-induced Ca2+ mobilization was inhibited when the treatment was preceded by wounding. Mechanical wounding promoted CREB phosphorylation, and the presence of MASP-1 enhanced this effect. Wounding induced ICAM-1 and VCAM-1 expression on endothelial cells, and MASP-1 pretreatment further increased VCAM-1 levels. MASP-1 played a role in the subsequent stages of angiogenesis, facilitating the breakdown of the endothelial capillary network on Matrigel®. Our findings extend our general understanding of endothelial wound healing and highlight the importance of complement MASP-1 activation in wound-healing processes.

PLK1 and its substrate MISP facilitate intrahepatic cholangiocarcinoma progression by promoting lymphatic invasion and impairing E-cadherin adherens junctions.

Intrahepatic cholangiocarcinoma (iCCA) is a subtype of CCA and has a high mortality rate and a relatively poor prognosis. However, studies focusing on increased cell motility and loss of epithelial integrity during iCCA progression remain relatively scarce. We collected seven fresh tumor samples from four patients to perform RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) to determine the transcriptome profile and chromatin accessibility of iCCA. The increased expression of cell cycle regulators, including PLK1 and its substrate MISP, was identified. Ninety-one iCCA patients were used to validate the clinical significance of PLK1 and MISP. The upregulation of PLK1 and MISP was determined in iCCA tissues. Increased expression of PLK1 and MISP was significantly correlated with tumor number, N stage, and lymphatic invasion in an iCCA cohort. Knockdown of PLK1 or MISP reduced trans-lymphatic endothelial migration and wound healing and affected focal adhesions in vitro. In cell‒cell junctions, MISP localized to adherens junctions and suppressed E-cadherin dimerization. PLK1 disrupted adherens junctions in a myosin-dependent manner. Furthermore, PLK1 and MISP promoted cell proliferation in vitro and tumorigenesis in vivo. In iCCA, PLK1 and MISP promote aggressiveness by increasing lymphatic invasion, tumor growth, and motility through the repression of E-cadherin adherens junctions.

Multi-Omics Analysis of Circulating Exosomes in Adherent Long-Term Treated OSA Patients.

Obstructive sleep apnea (OSA) is a highly prevalent chronic disease affecting nearly a billion people globally and increasing the risk of multi-organ morbidity and overall mortality. However, the mechanisms underlying such adverse outcomes remain incompletely delineated. Extracellular vesicles (exosomes) are secreted by most cells, are involved in both proximal and long-distance intercellular communication, and contribute toward homeostasis under physiological conditions. A multi-omics integrative assessment of plasma-derived exosomes from adult OSA patients prior to and after 1-year adherent CPAP treatment is lacking. We conducted multi-omic integrative assessments of plasma-derived exosomes from adult OSA patients prior to and following 1-year adherent CPAP treatment to identify potential specific disease candidates. Fasting morning plasma exosomes isolated from 12 adult patients with polysomnographically-diagnosed OSA were analyzed before and after 12 months of adherent CPAP therapy (mean ≥ 6 h/night) (OSAT). Exosomes were characterized by flow cytometry, transmission electron microscopy, and nanoparticle tracking analysis. Endothelial cell barrier integrity, wound healing, and tube formation were also performed. Multi-omics analysis for exosome cargos was integrated. Exosomes derived from OSAT improved endothelial permeability and dysfunction as well as significant improvement in tube formation compared with OSA. Multi-omic approaches for OSA circulating exosomes included lipidomic, proteomic, and small RNA (miRNAs) assessments. We found 30 differentially expressed proteins (DEPs), 72 lipids (DELs), and 13 miRNAs (DEMs). We found that the cholesterol metabolism (has04979) pathway is associated with lipid classes in OSA patients. Among the 12 subjects of OSA and OSAT, seven subjects had complete comprehensive exosome cargo information including lipids, proteins, and miRNAs. Multi-omic approaches identify potential signature biomarkers in plasma exosomes that are responsive to adherent OSA treatment. These differentially expressed molecules may also play a mechanistic role in OSA-induced morbidities and their reversibility. Our data suggest that a multi-omic integrative approach might be useful in understanding how exosomes function, their origin, and their potential clinical relevance, all of which merit future exploration in the context of relevant phenotypic variance. Developing an integrated molecular classification should lead to improved diagnostic classification, risk stratification, and patient management of OSA by assigning molecular disease-specific therapies.

D-2-hydroxyglutarate regulates human brain vascular endothelial cell proliferation and barrier function.

Gain-of-function mutations in isocitrate dehydrogenase (IDH) genes result in excessive production of (D)-2-hydroxyglutarate (D-2HG) which intrinsically modifies tumor cell epigenetics and impacts surrounding noncancerous cells through nonepigenetic pathways. However, whether D-2HG has a paracrine effect on endothelial cells in the tumor microenvironment needs further clarification. We quantified microvessel density by immunohistochemistry using tissue sections from 60 high-grade astrocytic gliomas with or without IDH mutation. Microvessel density was found to be reduced in tumors carrying an IDH mutation. Ex vivo experiments showed that D-2HG inhibited endothelial cell migration, wound healing, and tube formation by suppressing cell proliferation but not viability, possibly through reduced activation of the mTOR/STAT3 pathway. Further, D-2HG reduced fluorescent dextran permeability and decreased paracellular T-cell transendothelial migration by augmenting expression of junctional proteins thereby collectively increasing endothelial barrier function. These results indicate that D-2HG may influence the tumor vascular microenvironment by reducing the intratumoral vasculature density and by inhibiting the transport of metabolites and extravasation of circulating cells into the astrocytoma microenvironment. These observations provide a rationale for combining IDH inhibition with antitumor immunological/angiogenic approaches and suggest a molecular basis for resistance to antiangiogenic drugs in patients whose tumors express a mutant IDH allele.

ROCK Inhibitors as an Alternative Therapy for Corneal Grafting: A Systematic Review.

Purpose: Currently, corneal blindness is affecting >10 million individuals worldwide, and there is a significant unmet medical need because only 1.5% of transplantation needs are met globally due to a lack of high-quality grafts. In light of this global health disaster, researchers are developing corneal substitutes that can resemble the human cornea in vivo and replace human donor tissue. Thus, this review examines ROCK (Rho-associated coiled-coil containing protein kinases) inhibitors as a potential corneal wound-healing (CWH) therapy by reviewing the existing clinical and nonclinical findings. Methods: The systematic review was done from PubMed, Scopus, Web of Science, and Google Scholar for CWH, corneal injury, corneal endothelial wound healing, ROCK inhibitors, Fasudil, Netarsudil, Ripasudil, Y-27632, clinical trial, clinical study, case series, case reports, preclinical study, in vivo, and in vitro studies. After removing duplicates, all downloaded articles were examined. The literature search included the data till January 2023. Results: This review summarized the results of ROCK inhibitors in clinical and preclinical trials. In a clinical trial, various ROCK inhibitors improved CWH in individuals with open-angle glaucoma, cataract, iris cyst, ocular hypertension, and other ocular diseases. ROCK inhibitors also improved ocular wound healing by increasing cell adhesion, migration, and proliferation in vitro and in vivo. Conclusions: ROCK inhibitors have antifibrotic, antiangiogenic, anti-inflammatory, and antiapoptotic characteristics in CWH, according to the existing research. ROCK inhibitors were effective topical treatments for corneal infections. Ripasudil, Y-27632, H-1152, Y-39983, and AMA0526 are a few new ROCK inhibitors that may help CWH and replace human donor tissue.

Abstract 660: Injury Induced Connexin 43 Expression Plays A Role In Vascular Endothelial Wound Healing

Vascular surgeries, such as stent placement and coronary artery bypass, damage the endothelial lining of blood vessels. Impaired healing of endothelial damage is a primary contributor to vascular stent and bypass failure, putting patients at risk for myocardial infarction and death. As such, it is necessary to identify regulators of normal endothelial healing and understand how these factors are dysregulated when healing is impaired. Retrospective analysis of RNAseq data from a previous study investigating non-injured vs. regenerating endothelial cells (EC) in mouse aortas revealed a significant increase in the expression of connexin 43 (Cx43) throughout the EC healing process. Although Cx43 functions are important for skin wound healing, the role of connexin 43 in large artery endothelial wound healing has never been investigated. Here, we test the hypothesis that injury-induced increases in Cx43 expression and gap junction functions promote vascular endothelial wound healing. To study Cx43 in the regenerating endothelium in vivo, we developed a novel model of endothelial cell (EC) injury in mouse carotid arteries. In this model, carotids are ligated for a period of one minute, resulting in an EC denudation injury that heals over 24-hours. Western blotting of EC isolated from damaged carotids demonstrated an increase in Cx43 protein in injured mouse EC while immunofluorescent imaging revealed increases in Cx43 staining at EC borders indicative of increased gap junction signaling. Human coronary artery EC scratch assays were also used to study the process of endothelial wound repair in vitro. As Cx43 phosphorylation is the primary regulator of gap junction channel signaling, we used pharmacological strategies (novel peptide treatments) to increase protein kinase C (PKC) phosphorylation at Cx43 serine 368 (Cx43-pS368), associated with gap junction closure and reduced intracellular communication. In cultured EC, increasing Cx43-pS368 resulted in slower wound healing times, suggesting that limiting Cx43 gap junction communication impairs EC wound healing. In conclusion, our data indicate that injury-induced EC upregulation of Cx43 and increases in gap junction intracellular communication may play a key role in EC wound healing responses.

Donepezil prevents high glucose-induced endothelial barrier dysfunction and accelerates wound healing process in human coronary endothelial cells

Abstract The Cholinergic Anti-inflammatory Pathway (CAP) is a neurophysiological mechanism that regulates the immune system. Studies show that the CAP inhibits inflammation by suppressing cytokine synthesis via release of acetylcholine in the heart, lungs and gastrointestinal tract. Upon release, acetylcholine interacts with α7 nicotinic acetylcholine receptors (α7nAChR), down-regulate pro-inflammatory cytokine synthesis and prevent tissue damage and inflammation. Donepezil, a centrally acting reversible acetylcholinesterase inhibitor, has been used in the palliative treatment of Alzheimer’s disease (AD). We hypothesized that the donepezil prevents high glucose-induced endothelial barrier dysfunction and accelerates wound healing process in human coronary endothelial cells by preventing the destabilization of junctional proteins. We investigated the potential protective role of Donepezil in high glucose induced increase in human coronary artery endothelial (HCAE) permeability and wound healing process. Endothelial permeability was evaluated by using state of the art Electric Cell-substrate Impedance Sensing (ECIS) mechanism, a measure of trans-endothelial electrical resistance (TEER) across the endothelial monolayers using TEER electrodes. Wound healing assay was performed by employing electric signals to both wound and monitor the healing process in endothelial monolayer. Our data show that 25mM glucose concentration increased endothelial permeability and abrogated the wound healing process. Donepezil at the dose of 5 μM and 10 μM significantly prevented alteration of endothelial barrier function and enhances wound healing process in the presence of high glucose concentration.

Donepezil is emerging as a potential therapeutic treatment in improving endothelial barrier function to prevent and treat coronary artery disease in Diabetes Mellitus.

Abstract Current treatments have shown limited success in reversing DM induced vascular endothelial barrier dysfunction. Vascular endothelium forms the inner most lining of the blood vessels, regulates variety of biological processes such as angiogenesis, wound healing and host defense mechanisms. During inflammatory conditions, such as diabetes mellitus and myocardial infarction endothelial cell-cell junctions start to disrupt because of the internalization of the junctional proteins, such as vascular endothelial (VE) cadherin. This leads to the formation of minute inter-endothelial gaps, and the infiltration of protein-rich fluid and immune cells in the interstitial space. If remains unchecked, the persistent buildup of edema underlying the endothelial lining sets the stage for the serious life-threatening complications. Therefore, we hypothesized that the donepezil prevents high glucose-induced endothelial barrier dysfunction by preventing the destabilization of junctional proteins. We investigated the potential protective role of Donepezil in high glucose induced increase in human coronary artery endothelial (HCAE) permeability and wound healing process. Wound healing assay was performed by employing electric signals to both wound and monitor the healing process in endothelial monolayer. To investigate the signaling mechanism involved in the protection offered by Donepezil, we found that Donepezil prevented loss of VE-cadherin in endothelial junctions, and abrogated stress fiber formation in endothelial cell exposed to high-glucose solution. In conclusion, our study shows that Donepezil maintained endothelial barrier function and improved wound healing process in endothelial cells exposed to high glucose. Chicago State University CTRE grant to Professor Nadeem Fazal, MD, PhD

Mice Deficient in TAZ (Wwtr1) Demonstrate Clinical Features of Late-Onset Fuchs' Endothelial Corneal Dystrophy.

We sought to define the role of Wwtr1 in murine ocular structure and function and determine the role of mechanotransduction in Fuchs' endothelial corneal dystrophy (FECD), with emphasis on interactions between corneal endothelial cells (CEnCs) and Descemet's membrane (DM). A Wwtr1 deficient mouse colony was established, and advanced ocular imaging, atomic force microscope (AFM), and histology/immunofluorescence were performed. Corneal endothelial wound healing was assessed using cryoinjury and phototherapeutic keratectomy in Wwtr1 deficient mice. Expression of WWTR1/TAZ was determined in the corneal endothelium from normal and FECD-affected patients; WWTR1 was screened for coding sequence variants in this FECD cohort. Mice deficient in Wwtr1 had reduced CEnC density, abnormal CEnC morphology, softer DM, and thinner corneas versus wildtype controls by 2 months of age. Additionally, CEnCs had altered expression and localization of Na/K-ATPase and ZO-1. Further, Wwtr1 deficient mice had impaired CEnC wound healing. The WWTR1 transcript was highly expressed in healthy human CEnCs comparable to other genes implicated in FECD pathogenesis. Although WWTR1 mRNA expression was comparable between healthy and FECD-affected patients, WWTR1/TAZ protein concentrations were higher and localized to the nucleus surrounding guttae. No genetic associations were found in WWTR1 and FECD in a patient cohort compared to controls. There are common phenotypic abnormalities seen between Wwtr1 deficient and FECD-affected patients, suggesting that Wwtr1 deficient mice could function as a murine model of late-onset FECD. Despite the lack of a genetic association between FECD and WWTR1, aberrant WWTR1/TAZ protein subcellular localization and degradation may play critical roles in the pathogenesis of FECD.

Rock inhibitors and antisense oligonucleotides for Fuchs corneal endothelial dystrophy

AbstractFuchs corneal dystrophy (FECD) is the most common primary cause of corneal endothelial dystrophy. FECD is a complex disease where interaction between multiple genetic and environmental factors results in endothelial cells apoptosis and atypical extracellular matrix deposition. Due to the shortage of donor tissue, and high costs of transplants, a topical therapy for treating corneal endothelial dysfunction is urgently required. The topical treatment of several agents has been investigated as therapies for FECD, by the promotion of endothelial cell proliferation and migration, in experimental models of the disorder. ROCK1 and ROCK2 inhibition promotes cell adhesion, prevents apoptosis, and enhances the proliferation of and human corneal endothelial cells. These therapies have involved the knockdown of connexin by siRNA, the release of cell–cell contact inhibition with EDTA, and inhibition of Rho‐associated protein kinase (ROCK) with Y‐27632. Recently several papers showed that Ripasudil, a rho kinase inhibitor, has been shown to promote corneal endothelial wound healing in animal models and in clinical trials. An antisense oligonucleotide (ASO) has also been developed to target the repetitive RNA. This has been shown to decrease foci formation and reversal of misplacing in ex‐vivo FECD specimens. Antisense oligonucleotides (ASO) reduced the RNA aggregates and their toxic downstream events in patient cells. ASO has to be designed to target a particular genetic mutation. This new medical‐based regenerative therapy may provide a less invasive and more effective method for treating patients with FECD.

A passive pump-assisted microfluidic assay for quantifying endothelial wound healing in response to fluid shear stress.

There as an urgent need to quantify the endothelial wound-healing process in response to fluid shear stress to improve the biological and clinical understanding of healing mechanisms, which is of great importance for preventing healing impairment, chronic wounds, and postoperative in-stent restenosis. However, current experimental platforms not only require expensive, cumbersome, and powered pumping devices (to, e.g., generate cell scratches and load shear stress stimulation) but also lack quantitative controls for quantitative analysis. In this paper, a passive pump-assisted microfluidic assay is developed to quantify endothelial wound healing in response to fluid shear stress. Our assay consists of passive constant-flow pumps based on the siphon principle and a three-inlet microfluidic chip for cell wound-healing experiments. We also propose a method for quantitatively adjusting cell scratch size by controlling trypsin flow. Both numerical simulations and fluorescein experiments validate the effectiveness of this method. Moreover, we use the designed microfluidic assay to successfully generate cell scratches, load a 12-h shear stress of 5dyn/cm2 to the cells, and observe wound healing. The results indicate that the healing of a cell scratch is significantly accelerated under the stimulation of shear stress. In conclusion, our passive pump-assisted microfluidic assay shows versatility, applicability, and the potential for quantifying endothelial wound healing in response to fluid shear stress.

Comparative assessment of electronic nicotine delivery systems aerosol and cigarette smoke on endothelial cell migration: The Replica Project.

Cigarette smoking is associated with impairment of repair mechanisms necessary for vascular endothelium homeostasis. Reducing the exposure to smoke toxicants may result in the mitigation of the harmful effect on the endothelium and cardiovascular disease development. Previous investigations evaluated in vitro the effect of electronic cigarette (EC) compared with cigarette smoke demonstrating a significant reduction in human umbilical vein endothelial cells (HUVECs) migration inhibition following EC aerosol exposure. In the present study, we replicated one of these studies, evaluating the effects of cigarette smoke on endothelial cell migration compared with aerosol from EC and heated tobacco products (HTPs). We performed an in vitro scratch wound assay on endothelial cells with a multi-center approach (ring-study) to verify the robustness and reliability of the results obtained in the replicated study, also testing the effect of aerosol from two HTPs on endothelial cells. Consistently with the original study, we observed a substantial reduction of the effects of aerosol from EC and HTPs on endothelial cell migration compared with cigarette smoke. While cigarette smoke reduced endothelial wound healing ability already at low concentrations (12.5%) and in a concentration-dependent manner, EC and HTPs aerosol showed no effect on endothelial cells until 80%-100% concentrations. In conclusion, our study further confirms the importance of EC and tobacco heated products as a possible harm reduction strategy for cardiovascular diseases development in smokers.

Paneth Cells Regulate Lymphangiogenesis under Control of Microbial Signals during Experimental Portal Hypertension.

Intestinal microbiota can modulate portal hypertension through the regulation of the intestinal vasculature. We have recently demonstrated that bacterial antigens activate Paneth cells (PCs) to secrete products that regulate angiogenesis and portal hypertension. In the present work we hypothesized that Paneth cells regulate the development of lymphatic vessels under the control of intestinal microbiota during experimental portal hypertension. We used a mouse model of inducible PCs depletion (Math1Lox/LoxVilCreERT2) and performed partial portal vein ligation (PPVL) to induce portal hypertension. After 14 days, we performed mRNA sequencing and evaluated the expression of specific lymphangiogenic genes in small intestinal tissue. Intestinal and mesenteric lymphatic vessels proliferation was assessed by immunohistochemistry. Intestinal organoids with or without PCs were exposed to pathogen-associated molecular patterns, and conditioned media (CM) was used to stimulate human lymphatic endothelial cells (LECs). The lymphangiogenic activity of stimulated LECs was assessed by tube formation and wound healing assays. Secretome analysis of CM was performed using label-free proteomics quantification methods. Intestinal immune cell infiltration was evaluated by immunohistochemistry. We observed that the intestinal gene expression pattern was altered by the absence of PCs only in portal hypertensive mice. We found a decreased expression of specific lymphangiogenic genes in the absence of PCs during portal hypertension, resulting in a reduced proliferation of intestinal and mesenteric lymphatic vessels as compared to controls. In vitro analyses demonstrated that lymphatic tube formation and endothelial wound healing responses were reduced significantly in LECs treated with CM from organoids without PCs. Secretome analyses of CM revealed that PCs secrete proteins that are involved in lipid metabolism, cell growth and proliferation. Additionally, intestinal macrophages infiltrated the ileal mucosa and submucosa of mice with and without Paneth cells in response to portal hypertension. Our results suggest that intestinal microbiota signals stimulate Paneth cells to secrete factors that modulate the intestinal and mesenteric lymphatic vessels network during experimental portal hypertension.

IL-10 promotes endothelial progenitor cell infiltration and wound healing via STAT3.

Endothelial progenitor cells (EPCs) contribute tode novoangiogenesis, tissue regeneration, and remodeling. Interleukin 10 (IL-10), an anti-inflammatory cytokine that primarily signals via STAT3, has been shown to drive EPC recruitment to injured tissues. Our previous work demonstrated that overexpression of IL-10 in dermal wounds promotes regenerative tissue repair via STAT3-dependent regulation of fibroblast-specific hyaluronan synthesis. However, IL-10's role and specific mode of action on EPC recruitment, particularly in dermal wound healing and neovascularization in both normal and diabetic wounds, remain to be defined.Therefore, inducible skin-specific STAT3knockdown mice were studied to determine IL-10's impact on EPCs, dermal wound neovascularization and healing, and whether it is STAT3-dependent. We show that IL-10 overexpression significantly elevated EPC counts in the granulating wound bed, which was associated with robust capillary lumen density and enhanced re-epithelialization of both control and diabetic (db/db) wounds at day 7. We noted increased VEGF and high C-X-C motif chemokine 12 (CXCL12) levels in wounds and a favorable CXCL12gradient at day 3 that may support EPC mobilization and infiltration from bone marrow to wounds, an effect that was abrogated in STAT3knockdown wounds. These findings were supportedin vitro. IL-10 promoted VEGF and CXCL12synthesis in primary murine dermal fibroblasts, with blunted VEGF expression upon blocking CXCL12 in the media by antibody binding. IL-10-conditioned fibroblast media also significantly promoted endothelial sprouting and network formation. In conclusion, these studies demonstrate that overexpression of IL-10 in dermal wounds recruits EPCs and leads to increased vascular structures and faster re-epithelialization.

Microvesicles released from activated CD4+ T cells alter microvascular endothelial cell function.

BackgroundMicrovesicles are vesicles shed by plasma membranes following cell activation and apoptosis. The role of lymphocyte‐derived microvesicles in endothelial function remains poorly understood.MethodsCD4+ T cells isolated from peripheral blood of healthy human donors were stimulated using anti‐CD3/anti‐CD28‐coated beads. Proteomic profiling of microvesicles was performed using linear discriminant analysis (LDA) from activated T cells (MV.Act) and nonactivated T cells (MV.NAct). In addition, data processing analysis was performed using MaxQUANT workflow. Differentially expressed proteins found in MV.Act or MV.NAct samples with identification frequency = 100%, which were selected by both LDA (p < .01) and MaxQUANT (p < .01) workflows, were defined as “high‐confidence” differentially expressed proteins. Functional effects of MV.Act on human primary microvascular endothelial cells were analysed.ResultsT cells released large amounts of microvesicles upon stimulation. Proteomic profiling of microvesicles using LDA identified 2279 proteins (n = 2110 and n = 851 proteins in MV.Act and MV.NAct, respectively). Protein–protein interaction network models reconstructed from both differentially expressed proteins (n = 594; LDA p ≤ .01) and “high‐confidence” differentially expressed proteins (n = 98; p ≤ .01) revealed that MV.Act were enriched with proteins related to immune responses, protein translation, cytoskeleton organisation and TNFα‐induced apoptosis. For instance, MV.Act were highly enriched with IFN‐γ, a key proinflammatory pathway related to effector CD4+ T cells. Endothelial cell incubation with MV.Act induced superoxide generation, apoptosis, endothelial wound healing impairment and endothelial monolayer barrier disruption.ConclusionsT cell receptor‐mediated activation of CD4+ T cells stimulates the release of microvesicles enriched with proteins involved in immune responses, inflammation and apoptosis. T cell‐derived microvesicles alter microvascular endothelial function and barrier permeability, potentially promoting tissue inflammation.