Promote Endothelial Cells Proliferation Research Articles

Exploring the interactions between cardiomyocytes and endothelial cells to induce cardiac revascularization and regeneration

Abstract Funding Acknowledgements None. Background The adult heart is unable to regenerate after ischemic injury, primarily due to the incapacity of cardiomyocyte (CM) to re-enter in the cell cycle and endothelial cells (EC) to form new vessels. Interestingly, our recent data have unveiled that the decline of CM proliferation after birth is paralleled by a reduction in angiogenic potential. Nevertheless, the precise mechanisms governing angiogenesis inhibition in the adult heart remain elusive. We hypothesize that the terminal differentiation of CMs after birth leads to the paracrine suppression of EC proliferation. Purpose This study aims to identify membrane or secreted factors produced by adult CMs that inhibit EC proliferation and vessel formation in order to create new therapeutic strategies to induce cardiac revascularization and regeneration. Material and Methods To validate our hypothesis, ECs were co-cultured with CMs at distinct differentiation stages. Leveraging existing RNA sequencing datasets, we generated an interactome between ligands produced by CMs and receptors expressed by cardiac ECs. Through gain and loss of function studies, we identified pivotal anti-angiogenic factors and subsequently knocked-out the corresponding genes in Cas9 mice, with the aim of rescue the angiogenic potential of the adult heart. Concurrently, we assessed the ability of miR199a, a pro-proliferative miRNA known for inducing CM de-differentiation, to elicit EC proliferation and angiogenesis when delivered to the adult heart together with VEGF-A, utilizing adeno-associated viral vectors. Results Our findings revealed that adult CMs impaired EC proliferation. Comprehensive analysis identified 20 membrane and secreted factors, establishing 118 ligand-receptor interactions between CMs and ECs. Notably, KCNJ11 and CD74 emerged as key factors, as validated through gain and loss of function experiments. In vivo knockout of these genes in CMs led to significant increase in EC proliferation and the formation of neo-vessels. Furthermore, CM de-differentiation induced by miR199a demonstrated the capacity to promote EC proliferation and neo-vessel formation upon VEGF-A stimulation. Conclusions Collectively, this work demonstrated that fully mature CMs have a role in limiting angiogenic potential of the adult heart, likely explaining the failure of therapeutic angiogenesis approaches attempted so far. Our results suggest that innovative strategies targeting CM-EC cross-talk or inducing partial CM de-differentiation may open new therapeutic avenues in the field of cardiac regeneration.Adult CMs inhibit EC proliferationCMdedifferentiation rescues angiogenesis

Single-cell RNA sequencing analysis identifies one subpopulation of endothelial cells that proliferates and another that undergoes the endothelial-mesenchymal transition in regenerating pig hearts.

Background: In our previous work, we demonstrated that when newborn pigs undergo apical resection (AR) on postnatal day 1 (P1), the animals' hearts were completely recover from a myocardial infarction (MI) that occurs on postnatal day 28 (P28); single-nucleus RNA sequencing (snRNAseq) data suggested that this recovery was achieved by regeneration of pig cardiomyocyte subpopulations in response to MI. However, coronary vasculature also has a key role in promoting cardiac repair. Method: Thus, in this report, we used autoencoder algorithms to analyze snRNAseq data from endothelial cells (ECs) in the hearts of the same animals. Main results: Our results identified five EC clusters, three composed of vascular ECs (VEC1-3) and two containing lymphatic ECs (LEC1-2). Cells from VEC1 expressed elevated levels of each of five cell-cyclespecific markers (Aurora Kinase B [AURKB], Marker of Proliferation Ki-67 [MKI67], Inner Centromere Protein [INCENP], Survivin [BIRC5], and Borealin [CDCA8]), as well as a number of transcription factors that promote EC proliferation, while (VEC3 was enriched for genes that regulate intercellular junctions, participate in transforming growth factor β (TGFβ), bone morphogenic protein (BMP) signaling, and promote the endothelial mesenchymal transition (EndMT). The remaining VEC2 did not appear to participate directly in the angiogenic response to MI, but trajectory analyses indicated that it may serve as a reservoir for the generation of VEC1 and VEC3 ECs in response to MI. Notably, only the VEC3 cluster was more populous in regenerating (i.e., ARP1MIP28) than non-regenerating (i.e., MIP28) hearts during the 1-week period after MI induction, which suggests that further investigation of the VEC3 cluster could identify new targets for improving myocardial recovery after MI. Histological analysis of KI67 and EndMT marker PDGFRA demonstrated that while the expression of proliferation of endothelial cells was not significantly different, expression of EndMT markers was significantly higher among endothelial cells of ARP1MIP28 hearts compared to MIP28 hearts, which were consistent with snRNAseq analysis of clusters VEC1 and VEC3. Furthermore, upregulated secrete genes by VEC3 may promote cardiomyocyte proliferation via the Pi3k-Akt and ERBB signaling pathways, which directly contribute to cardiac muscle regeneration. Conclusion: In regenerative heart, endothelial cells may express EndMT markers, and this process could contribute to regeneration via a endothelial-cardiomyocyte crosstalk that supports cardiomyocyte proliferation.

Mechanistic insights into the anti-restenotic effects of HSP27 and HO1 modulated by reconstituted HDL on neointimal hyperplasia

High-density lipoprotein (HDL) therapy has demonstrated beneficial effects in acute stroke and acute myocardial infarction models by reducing infarct size. In this study, we investigated the inhibitory effects of reconstituted HDL (rHDL) on neointimal hyperplasia and elucidated its underlying mechanism using a balloon injury rat model. Our finding revealed a significant 37% reduction in the intima to media ratio in the arteries treated with 80 mg/kg rHDL compared to those subjected to injury alone (p < 0.05), indicating a specific inhibition of neointimal hyperplasia. In vivo analysis further supported the positive effects of rHDL by demonstrating a reduction in smooth muscle cell (SMC) proliferation and an increase in endothelial cell (EC) proliferation. Additionally, rHDL treatment led to decreased infiltration of leukocytes and downregulated the expression of matrix metallopeptidase 9 (MMP9) in the neointimal area. Notably, rHDL administration resulted in decreased expression of VCAM1 and HIF1α, alongside increased expression of heme oxygenase 1 (HO1) and heat shock protein 27 (HSP27). Overexpression of HSP27 and HO1 effectively inhibited SMC proliferation. Moreover, rHDL-mediated suppression of injury-induced HIF1α coincided with upregulation of HSP27. Interestingly, HSP27 and HO1 had varying effects on the expression of chemokine receptors and rHDL did not exert significant effect on chemokine receptor expression in THP1 cells. These findings underscore the distinct roles of HSP27 and HO1 as potential regulatory factors in the progression of restenosis. Collectively, our study demonstrates that rHDL exerts a potent anti-neointimal hyperplasia effect by reducing leukocytes infiltration and SMC proliferation while promoting EC proliferation.

Carbon monoxide release from ultrasound-sensitive microbubbles improves endothelial cell growth.

Carbon monoxide is a gasotransmitter that may be beneficial for vascular tissue engineering and regenerative medicine strategies because it can promote endothelial cell (EC) proliferation and migration by binding to heme-containing compounds within cells. For example, CO may be beneficial for vascular cognitive impairment and dementia because many patients' disrupted blood-brain barriers do not heal naturally. However, control of the CO dose is critical, and new controlled delivery methods need to be developed. This study developed ultrasound-sensitive microbubbles with a carefully controlled precipitation technique, loaded them with CO, and assessed their ability to promote EC proliferation and function. Microbubbles fabricated with perfluoropentane exhibited good stability at room temperature, but they could still be ruptured and release CO in culture with application of ultrasound. Microbubbles synthesized from the higher boiling point compound, perfluorohexane, were too stable at physiological temperature. The lower-boiling point perfluoropentane microbubbles had good biocompatibility and appeared to improve VE-cadherin expression when CO was loaded in the bubbles. Finally, tissue phantoms were used to show that an imaging ultrasound probe can efficiently rupture the microbubbles and that the CO-loaded microbubbles can improve EC spreading and proliferation compared to control conditions without microbubbles as well as microbubbles without application of ultrasound. Overall, this study demonstrated the potential for use of these ultrasound-sensitive microbubbles for improving blood vessel endothelialization.

Reduction in mitochondrial ROS improves oxidative phosphorylation and provides resilience to coronary endothelium in non-reperfused myocardial infarction.

Recent studies demonstrated that mitochondrial antioxidant MnSOD that reduces mitochondrial (mito) reactive oxygen species (ROS) helps maintain an optimal balance between sub-cellular ROS levels in coronary vascular endothelial cells (ECs). However, it is not known whether EC-specific mito-ROS modulation provides resilience to coronary ECs after a non-reperfused acute myocardial infarction (MI). This study examined whether a reduction in endothelium-specific mito-ROS improves the survival and proliferation of coronary ECs in vivo. We generated a novel conditional binary transgenic animal model that overexpresses (OE) mitochondrial antioxidant MnSOD in an EC-specific manner (MnSOD-OE). EC-specific MnSOD-OE was validated in heart sections and mouse heart ECs (MHECs). Mitosox and mito-roGFP assays demonstrated that MnSOD-OE resulted in a 50% reduction in mito-ROS in MHEC. Control and MnSOD-OE mice were subject to non-reperfusion MI surgery, echocardiography, and heart harvest. In post-MI hearts, MnSOD-OE promoted EC proliferation (by 2.4 ± 0.9 fold) and coronary angiogenesis (by 3.4 ± 0.9 fold), reduced myocardial infarct size (by 27%), and improved left ventricle ejection fraction (by 16%) and fractional shortening (by 20%). Interestingly, proteomic and Western blot analyses demonstrated upregulation in mitochondrial complex I and oxidative phosphorylation (OXPHOS) proteins in MnSOD-OE MHECs. These MHECs also showed increased mitochondrial oxygen consumption rate (OCR) and membrane potential. These findings suggest that mito-ROS reduction in EC improves coronary angiogenesis and cardiac function in non-reperfused MI, which are associated with increased activation of OXPHOS in EC-mitochondria. Activation of an energy-efficient mechanism in EC may be a novel mechanism to confer resilience to coronary EC during MI.

Activation of angiotensin II type 2 receptor attenuates lung injury of collagen-induced arthritis by alleviating endothelial cell injury and promoting Ly6Clo monocyte transition

As one of the most frequent extra-articular manifestations of rheumatoid arthritis (RA), interstitial lung disease (ILD) is still challenging due to unrevealed pathophysiological mechanism. To address this question, in the present study, we used the classical collagen-induced arthritis (CIA) mouse model to determine the related-immune mechanism of lung injury and possible pharmacological treatment for RA-ILD. At the peak of arthritis, we found CIA mice developed apparent lung injury, characterized by interstitial thickening, inflammatory cell infiltration, and lymphocyte follicle formation. Additionally, the endothelial injury occurred as the number of endothelial cells (ECs) and their CD31 expression decreased. Along with those, monocytes, predominantly Ly6Chi monocytes with pro-inflammatory phenotype, were also increased. While in the remission period of arthritis, ECs gradually increased with retrieved CD31 expression, leading to decreased infiltrating monocytes, but boosted Ly6Clo population. Ly6Clo monocytes were prone to locate around damaged ECs, promoted ECs proliferation and vascular tube formation, and lessened the expression of adhesion molecules. In addition, we evaluated angiotensin II type 2 receptor (Agtr2), which has been demonstrated to be protective against lung injury, could be beneficial in RA-ILD. We found elevated Agtr2 in CIA lung tissue, and activation of Agtr2, within its specific agonist C21, alleviated the pulmonary inflammation in vivo, reduced ECs injury, and promoted monocytes conversion from Ly6Chi to Ly6Clo monocytes in vitro. Our data reveal a potential pathological mechanism of RA-ILD that involves ECs damage and inflammatory monocytes infiltration and provide a potential drug target, Agtr2, for RA-ILD treatment.

Abstract P1028: The Epicardium As The Conductor Of Cardiovascular Cells In Cardiac Regeneration

Regenerative cardiovascular medicine is emerging as a promising therapeutic alternative to heart transplantation in patients suffering from chronic heart failure. Although promising, challenges to cell therapy for cardiac repair remain, including cell survival, maturation, graft size, revascularisation, and immunogenicity. It has recently been shown that co-transplantation of human embryonic stem cell (hESC)-derived epicardial cells (EPI) with hESC-derived cardiomyocytes (CM) improves cardiac repair processes with respect to engraftment, cell maturation, and graft and host vascularisation. One plausible explanation for the observed benefits of co-transplantation is paracrine-mediated effects. While RNA sequencing data has been used to identify putative players in hESC-EPIs, little is known about their involvement in mediating hESC-CM maturation and revascularization, in particular in communication with endothelial cells (EC). Here, the role of paracrine signalling in hESC-EPI-mediated promotion of hESC-CM maturation and angiogenesis is being investigated with an emphasis on the involvement of extracellular vesicles (EVs). hESC-EPI-EVs improved responsiveness to pacing and coordination of contraction in vitro in hESC-CM cell culture and engineered heart tissues (EHTs). Moreover, hESC-EPI-EVs and hESC-EPI supernatant excluding EVs independently promoted tube formation in in vitro gel-based angiogenesis assays. hESC-EPI supernatant excluding EVs also promoted EC proliferation in MTS assays and EC migration in wound closure assays. Characterisation of the hESC-EPI-EV-cargo on a protein level revealed candidate factors involved in cardiac repair processes. Together with hESC-EPI-EV miRNA-sequencing and hESC-EPI gene expression data key paracrine signalling pathways and candidate factors to manipulate them are being identified and investigated in in vitro angiogenesis and CM maturation assays. Promising angiogenesis promoting candidates are being validated in vivo using yolk-sac membrane and chorioallantoic membrane assays. Identification of the paracrine signalling pathways involved would make it possible to effectively address selected cardiac repair processes, resulting in new approaches to therapy.

Profilin 2 and Endothelial Exosomal Profilin 2 Promote Angiogenesis and Myocardial Infarction Repair in Mice.

Cardiovascular diseases (CVD) are the leading cause of death worldwide, wherein myocardial infarction (MI) is the most dangerous one. Promoting angiogenesis is a prospective strategy to alleviate MI. Our previous study indicated that profilin 2 (PFN2) may be a novel target associated with angiogenesis. Further results showed higher levels of serum PFN2 and exosomal PFN2 in patients, mice, and pigs with MI. In this study, we explored whether PFN2 and endothelial cell (EC)-derived exosomal PFN2 could increase angiogenesis and be beneficial for the treatment of MI. Serum PFN2, exosomes, and exosomal PFN2 were elevated in rats with MI. PFN2 and exosomes from PFN2-overexpressing ECs (OE-exo) enhanced EC proliferation, migration, and tube formation ability. OE-exo also significantly increased the vessel number in zebrafish and protected the ECs from inflammatory injury. Moreover, OE-exo-treated mice with MI showed improvement in motor ability, ejection fraction, left ventricular shortening fraction, and left ventricular mass, as well as increased vessel numbers in the MI location, and decreased infarction volume. Mechanistically, PI3K might be the upstream regulator of PFN2, while ERK might be the downstream regulator in the PI3K-PFN2-ERK axis. Taken together, our findings demonstrate that PFN2 and exosomal PFN2 promote EC proliferation, migration, and tube formation through the PI3K-PFN2-ERK axis. Exosomal PFN2 may be a valuable target in the repair of MI injury via angiogenesis.

Activated mesangial cells induce glomerular endothelial cells proliferation in rat anti-Thy-1 nephritis through VEGFA/VEGFR2 and Angpt2/Tie2 pathway.

ObjectivesWe aimed to investigate the underlying mechanism of endothelial cells (ECs) proliferation in anti‐Thy‐1 nephritis.Materials and methodsWe established anti‐Thy‐1 nephritis and co‐culture system to explore the underlying mechanism of ECs proliferation in vivo and in vitro. EdU assay kit was used for measuring cell proliferation. Immunohistochemical staining and immunofluorescence staining were used to detect protein expression. ELISA was used to measure the concentration of protein in serum and medium. RT‐qPCR and Western blot were used to qualify the mRNA and protein expression. siRNA was used to knock down specific protein expression.ResultsIn anti‐Thy‐1 nephritis, ECs proliferation was associated with mesangial cells (MCs)‐derived vascular endothelial growth factor A (VEGFA) and ECs‐derived angiopoietin2 (Angpt2). In vitro co‐culture system activated MCs‐expressed VEGFA to promote vascular endothelial growth factor receptor2 (VEGFR2) activation, Angpt2 expression and ECs proliferation, but inhibit TEK tyrosine kinase (Tie2) phosphorylation. MCs‐derived VEGFA stimulated Angpt2 expression in ECs, which inhibited Tie2 phosphorylation and promoted ECs proliferation. And decline of Tie2 phosphorylation induced ECs proliferation. In anti‐Thy‐1 nephritis, promoting Tie2 phosphorylation could alleviate ECs proliferation.ConclusionsOur study showed that activated MCs promoted ECs proliferation through VEGFA/VEGFR2 and Angpt2/Tie2 pathway in experimental mesangial proliferative glomerulonephritis (MPGN) and in vitro co‐culture system. And enhancing Tie2 phosphorylation could alleviate ECs proliferation, which will provide a new idea for MPGN treatment.

Inhibition of protein glycosylation is a novel pro-angiogenic strategy that acts via activation of stress pathways

Endothelial cell (EC) metabolism is thought to be one of the driving forces for angiogenesis. Here we report the identification of the hexosamine D-mannosamine (ManN) as an EC mitogen and survival factor for bovine and human microvascular EC, with an additivity with VEGF. ManN inhibits glycosylation in ECs and induces significant changes in N-glycan and O-glycan profiles. We further demonstrate that ManN and two N-glycosylation inhibitors stimulate EC proliferation via both JNK activation and the unfolded protein response caused by ER stress. ManN results in enhanced angiogenesis in a mouse skin injury model. ManN also promotes angiogenesis in a mouse hindlimb ischemia model, with accelerated limb blood flow recovery compared to controls. In addition, intraocular injection of ManN induces retinal neovascularization. Therefore, activation of stress pathways following inhibition of protein glycosylation can promote EC proliferation and angiogenesis and may represent a therapeutic strategy for treatment of ischemic disorders.

Increasing the expression of microRNA-126-5p in the temporal muscle can promote angiogenesis in the chronically ischemic brains of rats subjected to two-vessel occlusion plus encephalo-myo-synangiosis.

Background: miR-126-5p plays an important role in promoting endothelial cell (EC) proliferation. We thus explored whether miR-126-5p can promote EC proliferation and angiogenesis in chronically ischemic brains (CIBs).Results: Improved revascularization in moyamoya patients was correlated with upregulated miR-126-5p expression in the TM and DM. In vitro experiments showed that miR-126-5p promoted EC proliferation through the PI3K/Akt pathway. CIBs from the agomir group exhibited significantly higher p-Akt, VEGF, CD31 and eNOS expression compared with the control CIBs. The ICBP and the RCF were significantly better in the agomir compared with the control group.Conclusion: Increasing miR-126-5p expression in the TM can promote EC proliferation and angiogenesis in CIBs of 2VO+EMS rats through the PI3K/Akt pathway.Methods: We assessed the correlation between revascularization and miR-126-5p expression in the temporal muscle (TM) and dura mater (DM) of moyamoya patients. The effect of miR-126-5p on EC proliferation and downstream signaling pathways was explored in vitro. We established an animal model of two-vessel occlusion plus encephalo-myo-synangiosis (2VO+EMS), transfected the TM with miR-126-5p agomir/antagomir, compared the expression of miR-126-5p and relevant downstream cytokines in brain tissue among different groups, and investigated the improvement in cerebral blood perfusion (ICBP) and the recovery of cognitive function (RCF).

Hyaluronic acid oligosaccharide-modified collagen nanofibers as vascular tissue-engineered scaffold for promoting endothelial cell proliferation

Hyaluronic acid oligosaccharides (oHAs) have shown promising results in promoting vascular endothelial cell (EC) proliferation and endothelialization. To engineered develop tissue scaffold for promoting EC proliferation and vessel endothelialization, different sizes of oHAs were prepared and grafted onto collagen to improve the biological properties of the synthesized materials, especially in angiogenesis. Firstly, oHAs were successfully prepared and conjugated with collagen to construct glycosylated collagens by reductive amination. The glycosylated collagens were then electrospun into various nanofibrous structures and their morphology and hemocompatibility, as well as EC responses on these nanofibrous scaffolds, were studied to evaluate the potential for vascular tissue engineering. The results showed that the nanofibrous scaffolds grafted by oHAs promoted EC proliferation whereas high molecular weight HA inhibited proliferation. The scaffolds had no detectable degree of hemolysis and coagulation, suggesting their promise as engineered vascular tissue scaffolds.

Mussel adhesive protein fused with VE-cadherin extracellular domain promotes endothelial-cell tight junctions and in vivo endothelization recovery of vascular stent.

Improving the surface properties of vascular stents to accelerate endothelialization in vivo could play an important role in minimizing the risk of late thrombosis. We previously showed that mussel adhesive protein fused with VE-cadherin extracellular domain (VE-M) specifically triggered endothelial cell adhesion in vitro. In this study, using stent implants coated with VE-M, we evaluated the clinical applicability of VE-M in endothelialization recovery in vivo. First, we explored the effect of VE-M on hemocompatibility and tight junctions between endothelial cells (ECs) in vitro. VE-M significantly inhibited platelet adhesion and promoted EC proliferation. Furthermore, VE-M drastically increased the centralization of F-actin in human umbilical vein endothelial cells (HUVECs) along the cell contacts, reduced fluorescein isothiocyanate (FITC)-dextran transport across the HUVECs, and elevated expression levels of tight junction proteins (TJPs) in ECs. We then evaluated the effect of VE-M on endothelialization recovery in vivo through implantation of vascular stents. At 1 day after implantation, stents coated with VE-M recruited more endothelial progenitor cells (EPCs) than bare stents. At 7 days after implantation, VE-M stents had a greater coverage of ECs than bare stents. At 1 month after implantation, ECs on VE-M stents were appropriately elliptical in morphology and closely resembled physiological morphology. Hematoxylin-eosin (HE) staining revealed little in-stent neointima formation on VE-M stents, and SEM images revealed that smooth endothelium had formed on VE-M stents without adherent platelets. Taken together, these findings indicate that VE-M accelerates in vivo endothelialization of vascular stents via recruitment of EPCs and promotes endothelium formation and could be explored as a potential bioactive coating for vascular implant. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:94-103, 2020.

M2 macrophages promote pulmonary endothelial cells regeneration in sepsis-induced acute lung injury.

Macrophages can polarize to M2 phenotype to decrease inflammation and encourage tissue repair. Nonetheless, its role in sepsis-induced acute lung injury and its effect on endothelial cells (ECs) regeneration remains unknown. The aim of the current study was to explore the impact of M2 macrophages on pulmonary ECs proliferation in sepsis-induced acute lung injury. We co-cultured mouse lung microvascular endothelial cells (MLMVECs) with M2 macrophages following LPS challenge. M2 macrophages were intratracheally transplanted into mice subjected to cecal ligation and puncture (CLP). We further performed cytokine array for the supernatant from M2 macrophages and serum from mice subjected with CLP. We found both co-culture with M2 macrophages and treating with supernatant from M2 macrophages increased ECs viability following LPS challenge. Intratracheal transplantation of M2 macrophages markedly promoted pulmonary ECs proliferation, manifesting as attenuation of lung microvascular permeability and lung tissue edema, as well as improvement of survival rate. We further found that CXCL12, IL-1ra, TIMP-1, IL-4, and CXCL1 were increased in the supernatant of M2 macrophages in vitro. G-CSF and Complement Component 5a (C5/C5a) were increased in the serum of the M2-transplanted mice. The present study suggested M2 macrophages could promote ECs proliferation in sepsis-induced ALI through secretion of anti-inflammatory cytokines and growth factors.

Immobilization of nano Cu-MOFs with polydopamine coating for adaptable gasotransmitter generation and copper ion delivery on cardiovascular stents

In-stent restenosis is worsened by thrombosis, acute inflammation, and uncontrollable smooth muscle cells (SMCs) proliferation at the early stage of implantation. Tailoring the stent surface can inhibit thrombosis, intimal hyperplasia, and accelerate re-endothelialization. In situ nitric oxide (NO) generation is considered as a promising method to improve anti-coagulation and anti-hyperplasia abilities. Copper based metal organic frameworks showed great potential as catalysts for NO generation, and copper ion (Cu2+) was demonstrated to promote endothelial cells (ECs) growth. Herein, by using polydopamine as the linker and coating matrix, nanoscale copper-based metal organic frameworks (nano Cu-MOFs) were immobilized onto the titanium surface for simultaneous nitric oxide (NO) catalytic generation and Cu2+ delivery. The nano Cu-MOFs-immobilized coating exhibited desirable NO release and adaptable Cu2+ delivery. Such coating inhibited platelet aggregation and activation via NO-cGMP signaling pathway, and significantly reduced thrombosis in an ex vivo extracorporeal circulation model. NO release and Cu2+ delivery showed synergetic effect to promote EC proliferation. Moreover, SMCs and macrophage proliferation was suppressed by the nano Cu-MOFs-immobilized coating, thereby reducing neointimal hyperplasia in vivo. Overall, this biocompatible coating is convenient for the surface modification of cardiovascular stents and effectively prevents the late stent thrombosis and in-stent restenosis associated with stent implantation.

Smooth Muscle Contact Drives Endothelial Regeneration by BMPR2-Notch1-Mediated Metabolic and Epigenetic Changes.

Maintaining endothelial cells (EC) as a monolayer in the vessel wall depends on their metabolic state and gene expression profile, features influenced by contact with neighboring cells such as pericytes and smooth muscle cells (SMC). Failure to regenerate a normal EC monolayer in response to injury can result in occlusive neointima formation in diseases such as atherosclerosis and pulmonary arterial hypertension. We investigated the nature and functional importance of contact-dependent communication between SMC and EC to maintain EC integrity. We found that in SMC and EC contact cocultures, BMPR2 (bone morphogenetic protein receptor 2) is required by both cell types to produce collagen IV to activate ILK (integrin-linked kinase). This enzyme directs p-JNK (phospho-c-Jun N-terminal kinase) to the EC membrane, where it stabilizes presenilin1 and releases N1ICD (Notch1 intracellular domain) to promote EC proliferation. This response is necessary for EC regeneration after carotid artery injury. It is deficient in EC-SMC Bmpr2 double heterozygous mice in association with reduced collagen IV production, decreased N1ICD, and attenuated EC proliferation, but can be rescued by targeting N1ICD to EC. Deletion of EC- Notch1 in transgenic mice worsens hypoxia-induced pulmonary hypertension, in association with impaired EC regenerative function associated with loss of precapillary arteries. We further determined that N1ICD maintains EC proliferative capacity by increasing mitochondrial mass and by inducing the phosphofructokinase PFKFB3 (fructose-2,6-bisphosphatase 3). Chromatin immunoprecipitation sequencing analyses showed that PFKFB3 is required for citrate-dependent H3K27 acetylation at enhancer sites of genes regulated by the acetyl transferase p300 and by N1ICD or the N1ICD target MYC and necessary for EC proliferation and homeostasis. Thus, SMC-EC contact is required for activation of Notch1 by BMPR2, to coordinate metabolism with chromatin remodeling of genes that enable EC regeneration, and to maintain monolayer integrity and vascular homeostasis in response to injury.

TSPYL5-mediated inhibition of p53 promotes human endothelial cell function

Testis-specific protein, Y-encoded like (TSPYL) family proteins (TSPYL1-6), which are members of the nucleosome assembly protein superfamily, have been determined to be involved in the regulation of various cellular functions. However, the potential role of TSPYL family proteins in endothelial cells (ECs) has not been determined. Here, we demonstrated that the expression of TSPYL5 is highly enriched in human ECs such as human umbilical vein endothelial cells (HUVECs) and human pluripotent stem cell-differentiated ECs (hPSC-ECs). Importantly, TSPYL5 overexpression was shown to promote EC proliferation and functions, such as migration and tube formation, by downregulating p53 expression. Adriamycin-induced senescence was markedly blocked by TSPYL5 overexpression. In addition, the TSPYL5 depletion-mediated loss of EC functions was blocked by p53 inhibition. Significantly, TSPYL5 overexpression promoted angiogenesis in Matrigel plug and wound repair in a mouse skin wound healing model in vivo. Our results suggest that TSPYL5, a novel angiogenic regulator, plays a key role in maintaining endothelial integrity and function. These findings extend the understanding of TSPYL5-dependent mechanisms underlying the regulation of p53-related functions in ECs.

Invited commentary

Preventing the advancement of or, better yet, reversing peripheral vascular occlusive disease could save many limbs and provide a better quality of life for our patients. The “magic bullet” of an individual agent placed locally to change the milieu sufficiently to improve collateral vascular development or to stimulate angiogenesis has not been successful to date. However, providing cells that can change the local milieu by providing a slurry of beneficial agents is more promising. Mesenchymal stem cells (MSCs) can provide stromal support and paracrine effects of potential benefit. Unfortunately, one group of patients who could benefit most are those with diabetes, and yet their MSCs (dMSCs) have a deficient expansion capacity. This paper tests the hypothesis that dMSCs have a reversible defect in the epidermal growth factor (EGF) receptor pathway that may blunt cell growth and survival. Human platelet lysate (PL), known to be rich in several growth factors including EGF, might reverse this deficiency, as might EGF alone. It tests whether dMSCs can provide angiogenic stimulus of human umbilical vein endothelial cells (ECs) and, therefore, be a viable regenerative treatment. The study demonstrates that MSCs from the bone marrow and adipose tissue of limbs of well-controlled, nonsmoking diabetic patients have similar population doublings and entered senescence after passage 11, confirming deficient expansion capacity. In addition, dMSCs do not express EGF in their secretome while demonstrating robust expression of other angiogens. Supplementing the dMSCs with PL increased phosphorylation of AKT to healthy MSC levels, whereas providing EGF alone increased only phosphorylated extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) to similar levels as that of PL supplementation. MSC invasion increased over time, but there were inconsistent differences of invasion length and chemokine concentration across MSC types and patients. Both cell types were able to stimulate EC proliferation like standard EC growth media and in co-culture demonstrated longer EC sprouts. In contrast to past literature, this study confirms a lack of EGF expression by dMSCs, providing a deficiency that is cell culture modifiable. Adding EGF increased phosphorylated ERK1/2 levels but not pAKT levels, whereas PL increased both, which may be important because both AKT and ERK1/2 signaling pathways promote cell survival, proliferation, and sustained paracrine activity. The lack of EGF may explain the early senescence noted in the study, but exogenous EGF certainly does not fully correct deficient signaling pathways, whereas PL does better but likely not totally in considering prior studies by the same authors. Both types of dMSCs promote EC proliferation and sprouting, which supports the regenerative benefits of these cells if sufficient numbers for local injection are available. This study has limitations regarding sample size, potential lack of generalization (the population was nonsmoking, well-controlled diabetic patients), and potential bias because the control MSCs were from the iliac crest whereas the experimental MSCs were from the tibia. Provided is some detailed explanation as to how these limitations can be interpreted. Overall, this study is another step forward in the basic science wheel of evidence that will eventually lead to a clinical advancement in regenerative medicine aimed at limb salvage. The opinions or views expressed in this commentary are those of the author and do not necessarily reflect the opinions or recommendations of the Journal of Vascular Surgery the Society for Vascular Surgery. Reversible secretome and signaling defects in diabetic mesenchymal stem cells from peripheral arterial disease patientsJournal of Vascular SurgeryVol. 68Issue 6PreviewRegenerative medicine seeks to stall or to reverse the pathologic consequences of chronic diseases. Many people with diabetes have peripheral arterial disease (PAD), which increases their already high risk of major amputation. Cellular therapies are a promising regenerative medicine approach to PAD that can be used to focally inject regenerative cells to endangered tissue beds. Mesenchymal stem cells (MSCs) are known to promote tissue regeneration through stromal support and paracrine stimulation of new blood vessels (angiogenesis). Full-Text PDF Open Archive

Tetrahedral DNA Nanostructure Promotes Endothelial Cell Proliferation, Migration, and Angiogenesis via Notch Signaling Pathway

The problem of tissue vascularization is one of the obstacles that currently restricts the application of tissue engineering products to the clinic. Achieving tissue vascularization and providing adequate nutrients for tissues are an urgent problem to build complex and effective tissue-engineered tissues and organs. Therefore, the aim of this study was to investigate the effect of tetrahedral DNA nanostructures (TDNs), a novel and biocompatible nanomaterial, on angiogenesis. The results showed that TDNs can enter into endothelial cells (ECs) and promote EC proliferation, migration, tube formation, and expressions of angiogenic growth factors at the concentration of 250 nmol L-1, which was accompanied by activation of the Notch signaling pathway. These results provided a theoretical basis for the further understanding and potential use of TDNs in tissue engineering vascularization.

VEGF-A and VEGF-B Coordinate the Arteriogenesis to Repair the Infarcted Heart with Vagus Nerve Stimulation

Background/Aims: Vagus nerve stimulation (VNS) suppresses arrhythmic activity and minimizes cardiomyocyte injury. However, how VNS affects angiogenesis/arteriogenesis in infarcted hearts, is poorly understood. Methods: Myocardial infarction (MI) was achieved by ligation of the left anterior descending coronary artery (LAD) in rats. 7 days after LAD, stainless-steel wires were looped around the left and right vagal nerve in the neck for vagus nerve stimulation (VNS). The vagal nerve was stimulated with regular pulses of 0.2ms duration at 20 Hz for 10 seconds every minute for 4 hours, and then ACh levels by ELISA in cardiac tissue and serum were evaluated for its release after VNS. Three and 14 days after VNS, Real-time PCR, immunostaining and western blot were respectively used to determine VEGF-A/B expressions and α-SMA- and CD31-postive vessels in VNS-hearts with pretreatment of α7-nAChR blocker mecamylamine (10 mg/kg, ip) or mACh-R blocker atropine (10 mg/kg, ip) for 1 hour. The coronary function and left ventricular performance were analyzed by Langendorff system and hemodynamic parameters in VNS-hearts with pretreatment of VEGF-A/B-knockdown or VEGFR blocker AMG706. Coronary arterial endothelial cells proliferation, migration and tube formation were evaluated for angiogenesis following the stimulation of VNS in coronary arterial smooth muscle cells (VSMCs). Results: VNS has been shown to stimulate VEGF-A and VEGF-B expressions in coronary arterial smooth muscle cells (VSMCs) and endothelial cells (ECs) with an increase of α-SMA- and CD31-postive vessel number in infarcted hearts. The VNS-induced VEGF-A/B expressions and angiogenesis were abolished by m-AChR inhibitor atropine and α7-nAChR blocker mecamylamine in vivo. Interestingly, knockdown of VEGF-A by shRNA mainly reduced VNS-mediated formation of CD31⁺ microvessels. In contrast, knockdown of VEGF-B powerfully abrogated VNS-induced formation of α-SMA⁺ vessels. Consistently, VNS-induced VEGF-A showed a greater effect on EC tube formation as compared to VNS-induced VEGF-B. Moreover, VEGF-A promoted EC proliferation and VSMC migration while VEGF-B induced VSMC proliferation and EC migration in vitro. Mechanistically, vagal neurotransmitter acetylcholine stimulated VEGF-A/B expressions through m/nACh-R/PI3K/Akt/Sp1 pathway in EC. Functionally, VNS improved the coronary function and left ventricular performance. However, blockade of VEGF receptor by antagonist AMG706 or knockdown of VEGF-A or VEGF-B by shRNA significantly diminished the beneficial effects of VNS on ventricular performance. Conclusion: VNS promoted angiogenesis/arteriogenesis to repair the infracted heart through the synergistic effects of VEGF-A and VEGF-B.