GLP-1RA therapy increases circulating vascular regenerative cell content in people living with type 2 diabetes.

Glucagon-like peptide-1 receptor agonists (GLP-1RAs) and sodium-glucose cotransporter-2 (SGLT2) inhibitors are guideline-recommended therapies for the management of type 2 diabetes (T2D), atherosclerotic cardiovascular disease, heart failure, and chronic kidney disease. We previously observed in people living with T2D and coronary artery disease that circulating vascular regenerative (VR) progenitor cell content increased following 6-mo use of the SGLT2 inhibitor empagliflozin. In this post hoc subanalysis of the ORIGINS-RCE CardioLink-13 study (ClinicalTrials.gov Identifier NCT05253521), we analyzed the circulating VR progenitor cell content of 92 individuals living with T2D, among whom 20 were on a GLP-1RA, 42 were on an SGLT2 inhibitor but not a GLP-1RA, and 30 were on neither of these vascular protective therapies. In the GLP-1RA group, the mean absolute count of circulating VR progenitor cells defined by high aldehyde dehydrogenase (ALDH) activity (ALDHhiSSClow) and VR progenitor cells further characterized by surface expression of the proangiogenic marker CD133 (ALDHhiSSClowCD133+) was higher than the group receiving neither a GLP-1RA nor an SGLT2 inhibitor (P = 0.02) and comparable with that in the SGLT2 inhibitor group (P = 0.25). The absolute count of proinflammatory, granulocyte-restricted precursor cells (ALDHhiSSChi) was significantly lower in the GLP-1RA group compared with the group on neither therapy (P = 0.031). Augmented vessel repair initiated by VR cells with previously documented proangiogenic activity, alongside a reduction in systemic, granulocyte precursor-driven inflammation, may represent novel mechanisms responsible for the cardiovascular-metabolic benefits of GLP-1RA therapy. Prospective, randomized clinical trials are now warranted to establish the value of recovering circulating VR progenitor cell content with blood vessel regenerative functions.NEW & NOTEWORTHY In this post hoc subanalysis of 92 individuals living with T2D and at high cardiovascular risk, the authors summarize the differences in circulating vascular regenerative (VR) progenitor cell content between those on GLP-1RA therapy, on SGLT2 inhibitor without GLP-1RA therapy, and on neither therapy. Those on GLP-1RA therapy demonstrated greater circulating VR progenitor cell content and reduced proinflammatory granulocyte precursor content. These results offer novel mechanistic insights into the cardiometabolic benefits associated with GLP-1RA therapy.

Autophagy as a Guardian of Vascular Niche Homeostasis.

The increasing burden of vascular dysfunction on healthcare systems worldwide results in higher morbidity and mortality rates across pathologies, including cardiovascular diseases. Vasculopathy is suggested to be caused by the dysregulation of vascular niches, a microenvironment of vascular structures comprising anatomical structures, extracellular matrix components, and various cell populations. These elements work together to ensure accurate control of the vascular network. In recent years, autophagy has been recognized as a crucial regulator of the vascular microenvironment responsible for maintaining basic cell functions such as proliferation, differentiation, replicative senescence, and apoptosis. Experimental studies indicate that autophagy activation can be enhanced or inhibited in various pathologies associated with vascular dysfunction, suggesting that autophagy plays both beneficial and detrimental roles. Here, we review and assess the principles of autophagy organization and regulation in non-tumor vascular niches. Our analysis focuses on significant figures in the vascular microenvironment, highlighting the role of autophagy and summarizing evidence that supports the systemic or multiorgan nature of the autophagy effects. Finally, we discuss the critical organizational and functional aspects of the vasculogenic niche, specifically in relation to autophagy. The resulting dysregulation of the vascular microenvironment contributes to the development of vascular dysfunction.

Neural and Glial Regulation of Angiogenesis in CNS in Ischemic Stroke.

CNS diseases associated with compromised blood supply and/or vascular integrity are one of the leading causes of mortality and disability in adults worldwide and are also among 10 most common causes of death in children. Angiogenesis is an essential element of regeneration processes upon nervous tissue damage and can play a crucial role in neuroprotection. Here we review the features of cerebral vascular regeneration after ischemic stroke, including the complex interactions between endothelial cells and other brain cell types (neural stem cells, astrocytes, microglia, and oligodendrocytes). The mechanisms of reciprocal influence of angiogenesis and neurogenesis, the role of astrocytes in the formation of the blood-brain barrier, and roles of microglia and oligodendrocytes in vascular regeneration are discussed. Understanding the mechanisms of angiogenesis regulation in CNS is of critical importance for the development of new treatments of neurovascular pathologies.

Aging impairs the ability of vascular endothelial stem cells to generate endothelial cells in mice.

Tissue-resident vascular endothelial stem cells (VESCs), marked by expression of CD157, possess long-term repopulating potential and contribute to vascular regeneration and homeostasis in mice. Stem cell exhaustion is regarded as one of the hallmarks of aging and is being extensively studied in several types of tissue-resident stem cells; however, how aging affects VESCs has not been clarified yet. In the present study, we isolated VESCs from young and aged mice to compare their potential to differentiate into endothelial cells in vitro and in vivo. Here, we report that the number of liver endothelial cells (ECs) including VESCs was lower in aged (27-28 month-old) than young (2-3 month-old) mice. In vitro culture of primary VESCs revealed that the potential to generate ECs is impaired in aged VESCs isolated from liver and lung relative to young VESCs. Orthotopic transplantation of VESCs showed that aged VESCs and their progeny expand less efficiently than their young counterparts when transplanted into aged mice, but they are equally functional in young recipients. Gene expression analysis indicated that inflammatory signaling was more activated in aged ECs including VESCs. Using single-cell RNA sequencing data from the Tabula Muris Consortium, we show that T cells and monocyte/macrophage lineage cells including Kupffer cells are enriched in the aged liver. These immune cells produce IL-1ß and several chemokines, suggesting the possible involvement of age-associated inflammation in the functional decline of VESCs with age.

A coherent feed-forward loop drives vascular regeneration in damaged aerial organs of plants growing in a normal developmental context.

Aerial organs of plants, being highly prone to local injuries, require tissue restoration to ensure their survival. However, knowledge of the underlying mechanism is sparse. In this study, we mimicked natural injuries in growing leaves and stems to study the reunion between mechanically disconnected tissues. We show that PLETHORA (PLT) and AINTEGUMENTA (ANT) genes, which encode stem cell-promoting factors, are activated and contribute to vascular regeneration in response to these injuries. PLT proteins bind to and activate the CUC2 promoter. PLT proteins and CUC2 regulate the transcription of the local auxin biosynthesis gene YUC4 in a coherent feed-forward loop, and this process is necessary to drive vascular regeneration. In the absence of this PLT-mediated regeneration response, leaf ground tissue cells can neither acquire the early vascular identity marker ATHB8, nor properly polarise auxin transporters to specify new venation paths. The PLT-CUC2 module is required for vascular regeneration, but is dispensable for midvein formation in leaves. We reveal the mechanisms of vascular regeneration in plants and distinguish between the wound-repair ability of the tissue and its formation during normal development.

Impact Assessment of Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) and Hemostatic Sponge on Vascular Anastomosis Regeneration in Rats.

The proper regeneration of vessel anastomoses in microvascular surgery is crucial for surgical safety. Pituitary adenylate cyclase-activating polypeptide (PACAP) can aid healing by decreasing inflammation, apoptosis and oxidative stress. In addition to hematological and hemorheological tests, we examined the biomechanical and histological features of vascular anastomoses with or without PACAP addition and/or using a hemostatic sponge (HS). End-to-end anastomoses were established on the right femoral arteries of rats. On the 21st postoperative day, femoral arteries were surgically removed for evaluation of tensile strength and for histological and molecular biological examination. Effects of PACAP were also investigated in tissue culture in vitro to avoid the effects of PACAP degrading enzymes. Surgical trauma and PACAP absorption altered laboratory parameters; most notably, the erythrocyte deformability decreased. Arterial wall thickness showed a reduction in the presence of HS, which was compensated by PACAP in both the tunica media and adventitia in vivo. The administration of PACAP elevated these parameters in vitro. In conclusion, the application of the neuropeptide augmented elastin expression while HS reduced it, but no significant alterations were detected in collagen type I expression. Elasticity and tensile strength increased in the PACAP group, while it decreased in the HS decreased. Their combined use was beneficial for vascular regeneration.

Ion channels in stem cells and their roles in stem cell biology and vascular diseases.

Stem cell therapy may be a promising option for the treatment of vascular diseases. In recent years, significant progress has been made in stem cell research, especially in the mechanism of stem cell activation, homing and differentiation in vascular repair and reconstruction. Current research on stem cells focuses on protein expression and transcriptional networks. Ion channels are considered to be the basis for the generation of bioelectrical signals, which control the proliferation, differentiation and migration of various cell types. Although heterogeneity of multiple ion channels has been found in different types of stem cells, it is unclear whether the heterogeneous expression of ion channels is related to different cell subpopulations and/or different stages of the cell cycle. There is still a long way to go in clinical treatment by using the regulation of stem cell ion channels. In this review, we reviewed the main ion channels found on stem cells, their expression and function in stem cell proliferation, differentiation and migration, and the research status of stem cells' involvement in vascular diseases.

MFGE8 mitigates brain injury in a rat model of SAH by maintaining vascular endothelial integrity via TIGß5/PI3K/CXCL12 signaling.

Leaked blood components, injured endothelial cells, local inflammatory response and vasospasm may converge to promote microthrombosis following subarachnoid hemorrhage (SAH). Previously, we showed that the milk fat globule-epidermal growth factor 8 (MFGE8) can mitigate SAH-induced microthrombosis. This present study was aimed to explore the molecular pathway participated in MFGE8-dependent protection on vascular endothelium. Immunofluorescence, immunoblot and behavioral tests were used to determine the molecular partner and signaling pathway mediating the effect of MFGE8 in vascular endothelium in rats with experimental SAH and controls, together with the applications of RNA silencing and pharmacological intervention methods. Relative to control, recombinant human MFGE8 (rhMFGE8) treatment increased 5-bromo-2'-deoxyuridine (BrdU) labeled new endothelial cells, reduced TUNUL-positive endothelial cells and elevated the expression of phosphatidylinositol 3-kinase (PI3K) and chemokine (C-X-C motif) ligand 12 (CXCL12), in the brains of SAH rats. These effects were reversed by MFGE8 RNA silencing, as well as following cilengitide and wortmannin intervention. These results suggest that MFGE8 promotes endothelial regeneration and mitigates endothelial DNA damage through the activation of the TIGß5/PI3K/CXCL12 signaling pathway.

Diabetic pre-programming of myelopoiesis impairs tissue repair.

Bone marrow-derived monocyte-macrophages promote healing of injured tissue cooperatively with vasculogenic hematopoietic stem/progenitor cells. However, diabetes dysregulates hematopoiesis and attenuates bone marrow-derived tissue-reparative responses. In a recent issue of The Journal of Pathology, Barman et al extensively characterized myelopoietic responses in bone marrow following skin wounding in a type 2 model of diabetes. The study demonstrated that accumulation of monocyte-macrophages in the peripheral tissues is increased due to diabetic myelopoiesis that would oppose the reparative process following tissue injury. Interestingly, in this model, pathological myelopoiesis is independent of IL-1ß. The potential prophylactic and therapeutic implications of these data are discussed in terms of paracrine signaling, macrophage polarization, and hematopoietic stem cell mobilization/retention. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Transcriptional dynamics of pluripotent stem cell-derived endothelial cell differentiation revealed by single-cell RNA sequencing.

AIMS: Pluripotent stem cell-derived endothelial cell products possess therapeutic potential in ischaemic vascular disease. However, the factors that drive endothelial differentiation from pluripotency and cellular specification are largely unknown. The aims of this study were to use single-cell RNA sequencing (scRNA-seq) to map the transcriptional landscape and cellular dynamics of directed differentiation of human embryonic stem cell-derived endothelial cells (hESC-EC) and to compare these cells to mature endothelial cells from diverse vascular beds. METHODS AND RESULTS: A highly efficient directed 8-day differentiation protocol was used to generate a hESC-derived endothelial cell product (hESC-ECP), in which 66% of cells co-expressed CD31 and CD144. We observed largely homogeneous hESC and mesodermal populations at Days 0 and 4, respectively, followed by a rapid emergence of distinct endothelial and mesenchymal populations. Pseudotime trajectory identified transcriptional signatures of endothelial commitment and maturation during the differentiation process. Concordance in transcriptional signatures was verified by scRNA-seq analysis using both a second hESC line RC11, and an alternative hESC-EC differentiation protocol. In total, 105 727 cells were subjected to scRNA-seq analysis. Global transcriptional comparison revealed a transcriptional architecture of hESC-EC that differs from freshly isolated and cultured human endothelial cells and from organ-specific endothelial cells. CONCLUSION: A transcriptional bifurcation into endothelial and mesenchymal lineages was identified, as well as novel transcriptional signatures underpinning commitment and maturation. The transcriptional architecture of hESC-ECP was distinct from mature and foetal human EC.

In Situ Preconditioning of Human Mesenchymal Stem Cells Elicits Comprehensive Cardiac Repair Following Myocardial Infarction.

Human bone marrow-derived mesenchymal stem cells (BM-MSCs), represented as a population of adult stem cells, have long been considered as one of the most promising sources for cell-based cardiac regenerative therapy. However, their clinical use has been significantly hampered by low survival and poor retention following administration into failing hearts. Here, to improve the therapeutic effectiveness of BM-MSCs, we examined a novel therapeutic platform named in situ preconditioning in a rat myocardial infarction (MI) model. In situ preconditioning was induced by a combinatory treatment of BM-MSCs with genetically engineered hepatocyte growth factor-expressing MSCs (HGF-eMSCs) and heart-derived extracellular matrix (hdECM) hydrogel. Subsequently, our results demonstrated that in situ preconditioning with cell mixture substantially improved the survival/retention of BM-MSCs in the MI-induced rat hearts. Enhanced retention of BM-MSCs ultimately led to a significant cardiac function improvement, which was derived from the protection of myocardium and enhancement of vessel formation in the MI hearts. The results provide compelling evidence that in situ preconditioning devised to improve the therapeutic potential of BM-MSCs can be an effective strategy to achieve cardiac repair of MI hearts.

The biology of the extracorporeal vasculature of Botryllus schlosseri.

The extracorporeal vasculature of the colonial ascidian Botryllus schlosseri plays a key role in several biological processes: transporting blood, angiogenesis, regeneration, self-nonself recognition, and parabiosis. The vasculature also interconnects all individuals in a colony and is composed of a single layer of ectodermally-derived cells. These cells form a tube with the basal lamina facing the lumen, and the apical side facing an extracellular matrix that consists of cellulose and other proteins, known as the tunic. Vascular tissue is transparent and can cover several square centimeters, which is much larger than any single individual within the colony. It forms a network that ramifies and expands to the perimeter of each colony and terminates into oval-shaped protrusions known as ampullae. Botryllus individuals replace themselves through a weekly budding cycle, and vasculature is added to ensure the interconnection of each new individual, thus there is continuous angiogenesis occurring naturally. The vascular tissue itself is highly regenerative; surgical removal of the ampullae and peripheral vasculature triggers regrowth within 24-48 h, which includes forming new ampullae. When two individuals, whether in the wild or in the lab, come into close contact and their ampullae touch, they can either undergo parabiosis through anastomosing vessels, or reject vascular fusion. The vasculature is easily manipulated by direct means such as microinjections, microsurgeries, and pharmacological reagents. Its transparent nature allows for in vivo analysis by bright field and fluorescence microscopy. Here we review the techniques and approaches developed to study the different biological processes that involve the extracorporeal vasculature.

Stabilization of myeloid-derived HIFs promotes vascular regeneration in retinal ischemia.

The retinal vasculature is tightly organized in a structure that provides for the high metabolic demand of neurons while minimizing interference with incident light. The adverse impact of retinal vascular insufficiency is mitigated by adaptive vascular regeneration but exacerbated by pathological neovascularization. Aberrant growth of neovessels in the retina is responsible for impairment of sight in common blinding disorders including retinopathy of prematurity, proliferative diabetic retinopathy, and age-related macular degeneration. Myeloid cells are key players in this process, with diverse roles that can either promote or protect against ocular neovascularization. We have previously demonstrated that myeloid-derived VEGF, HIF1, and HIF2 are not essential for pathological retinal neovascularization. Here, however, we show by cell-specific depletion of Vhl in a mouse model of retinal ischemia (oxygen-induced retinopathy, OIR) that myeloid-derived HIFs promote VEGF and bFGF expression and enhance vascular regeneration in association with improved density and organization of the astrocytic network.

UTX/KDM6A Deletion Promotes Recovery of Spinal Cord Injury by Epigenetically Regulating Vascular Regeneration.

The regeneration of the blood vessel system post spinal cord injury (SCI) is essential for the repair of neurological function. As a significant means to regulate gene expression, epigenetic regulation of angiogenesis in SCI is still largely unknown. Here, we found that Ubiquitously Transcribed tetratricopeptide repeat on chromosome X (UTX), the histone H3K27 demethylase, increased significantly in endothelial cells post SCI. Knockdown of UTX can promote the migration and tube formation of endothelial cells. The specific knockout of UTX in endothelial cells enhanced angiogenesis post SCI accompanied with improved neurological function. In addition, we found regulation of UTX expression can change the level of microRNA 24 (miR-24) in vitro. The physical binding of UTX to the promotor of miR-24 was indicated by chromatin immunoprecipitation (ChIP) assay. Meanwhile, methylation sequencing of endothelial cells demonstrated that UTX could significantly decrease the level of methylation in the miR-24 promotor. Furthermore, miR-24 significantly abolished the promoting effect of UTX deletion on angiogenesis in vitro and in vivo. Finally, we predicted the potential target mRNAs of miR-24 related to angiogenesis. We indicate that UTX deletion can epigenetically promote the vascular regeneration and functional recovery post SCI by forming a regulatory network with miR-24.

Novel Cell-Based and Tissue Engineering Approaches for Induction of Angiogenesis as an Alternative Therapy for Diabetic Retinopathy.

Diabetic retinopathy (DR) is the most frequent microvascular complication of long-term diabetes and the most common cause of blindness, increasing morbidity in the working-age population. The most effective therapies for these complications include laser photocoagulation and anti-vascular endothelial growth factor (VEGF) intravitreal injections. However, laser and anti-VEGF drugs are untenable as a final solution as they fail to address the underlying neurovascular degeneration and ischemia. Regenerative medicine may be a more promising approach, aimed at the repair of blood vessels and reversal of retinal ischemia. Stem cell therapy has introduced a novel way to reverse the underlying ischemia present in microvascular complications in diseases such as diabetes. The present review discusses current treatments, their side effects, and novel cell-based and tissue engineering approaches as a potential alternative therapeutic approach.

MicroRNA-212 promotes the recovery function and vascular regeneration of endothelial progenitor cells in mice with ischemic stroke through inactivation of the notch signaling pathway via downregulating MMP9 expression.

Ischemic stroke is a refractory disease caused by cerebral ischemic injury, which results in brain dysfunction. This study intends to investigate the effects of microRNA-212 (miR-212) on the recovery function and vascular regeneration of endothelial progenitor cells (EPCs) by inactivation of the Notch signaling pathway by binding to matrix metallopeptidase 9 (MMP9) in mice with ischemic stroke. According to the results of database retrieval systems and data analysis, MMP9 was predicted as a gene related to ischemic stroke and miR-212 is a potential regulating mRNA of MMP9. All 72 healthy adult C57BL6 mice were selected for middle cerebral artery occlusion (MCAO) establishment. Cerebral infarction was observed under triphenyltetrazolium chloride staining. A series of inhibitors, activators, and siRNAs were introduced to the verified regulatory functions for miR-212 governing MMP9 in ischemic stroke. Cell proliferation was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and tube-forming ability by tubule formation test. Reverse transcription quantitative polymerase chain reaction and Western blot analysis were used to detect the expressions of miR-212, MMP9, Hes-1, and Notch-1. The corresponding results demonstrated that the area of cerebral infarction and the number of neuronal necrosis increased in the MCAO group in contrast to the sham group. Meanwhile, upregulation of miR-212 or downregulation of MMP9 decreases the expressions of MMP9, Hes-1 Notch-1, increases cell proliferation and tube-forming ability and improves the pathological conditions of EPCs. Our study suggests that miR-212 promotes recovery function and vascular regeneration of EPCs through negative regulation of the Notch signaling pathway via downregulating expression of MMP9, thus provides a clinical theoretical basis for ischemic stroke therapy.

Isolating and expanding endothelial progenitor cells from peripheral blood on peptide-functionalized polystyrene surfaces.

The expansion of human peripheral blood endothelial progenitor cells to obtain therapeutically relevant endothelial colony-forming cells (ECFCs) has been commonly performed on xeno-derived extracellular matrix proteins. For cellular therapy applications, xeno-free culture conditions are desirable to improve product safety and reduce process variability. We have previously described a novel fluorophore-tagged RGD peptide (RGD-TAMRA) that enhanced the adhesion of mature endothelial cells in vitro. To investigate whether this peptide can replace animal-derived extracellular matrix proteins in the isolation and expansion of ECFCs, peripheral blood mononuclear cells from 22 healthy adult donors were seeded on RGD-TAMRA-modified polystyrene culture surfaces. Endothelial colony formation was significantly enhanced on RGD-TAMRA-modified surfaces compared to the unmodified control. No phenotypic differences were detected between ECFCs obtained on RGD-TAMRA compared to ECFCs obtained on rat-tail collagen-coated surfaces. Compared with collagen-coated surfaces and unmodified surfaces, RGD-TAMRA surfaces promoted ECFC adhesion, cell spreading, and clonal expansion. This study presents a platform that allows for a comprehensive in vitro evaluation of peptide-based biofunctionalization as a promising avenue for ex vivo ECFC expansion.

microRNA-212-induced protection of the heart against myocardial infarction occurs via the interplay between AQP9 and PI3K/Akt signaling pathway.

Myocardial infarction (MI) is defined as the irreversible death of heart muscle that occurs secondary to prolonged lack of oxygen supply, which has resulted in millions of death worldwide. This study was conducted with aims of investigating how microRNA-212 (miR-212) and the inhibition of aquaporin-9 (AQP9) through the activation of the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling pathway plays a role in the prevention of MI. The relationship between miR-212 and AQP9 was determined with the use of bioinformatics combined with the dual luciferase reporter gene assay. Next, the MI model was established and the cardiomyocytes were transfected with different mimic, inhibitor and siRNAs to investigate the specific activity of miR-212, AQP9 and the PI3K/Akt signaling pathway in MI. The expression of miR-212, AQP9, PI3K, Akt, VEGF, Bax, and B cell lymphoma 2 (Bcl-2), along with cell apoptosis were determined using reverse transcription quantitative polymerase chain reaction (RT-qPCR), Western blot analysis and flow cytometry. Based on the results, AQP9 was verified as the direct target gene of miR-212. MI led to a decrease in miR-212 expression and an increase in AQP9 expression. It was also found that miR-212 activated the PI3K/Akt signaling pathway in MI through the inhibition of AQP9 expression. The overexpression of miR-212 or silencing AQP9 decreased cardiomyocytes apoptosis. These findings indicated that the overexpression of miR-212 inhibited AQP9 by activating the PI3K/Akt signaling pathway, thus decreasing cardiomyocytes apoptosis, promoting vascular regeneration and alleviating ventricular remodeling in rats with MI.

Enzyme-Aided Extraction of Fucoidan by AMG Augments the Functionality of EPCs through Regulation of the AKT/Rheb Signaling Pathway.

The purpose of the present study is to improve the endothelial progenitor cells (EPC) activation, proliferation, and angiogenesis using enzyme-aided extraction of fucoidan by amyloglucosidase (EAEF-AMG). Enzyme-aided extraction of fucoidan by AMG (EAEF-AMG) significantly increased EPC proliferation by reducing the reactive oxygen species (ROS) and decreasing apoptosis. Notably, EAEF-AMG treated EPCs repressed the colocalization of TSC2/LAMP1 and promoted perinuclear localization of mTOR/LAMP1 and mTOR/Rheb. Moreover, EAEF-AMG enhanced EPC functionalities, including tube formation, cell migration, and wound healing via regulation of AKT/Rheb signaling. Our data provided cell priming protocols to enhance therapeutic applications of EPCs using bioactive compounds for the treatment of CVD.