Human Dermal Microvascular Endothelial Cells Research Articles

The miR-590-3p/VEGFA axis modulates secretion of VEGFA from adipose-derived stem cells, which acts as a paracrine regulator of human dermal microvascular endothelial cell angiogenesis.

The paracrine secretion of angiogenic cytokines from adipose-derived stem cells (ADSCs) might promote endothelial cell angiogenesis, therefore promoting wound healing in injured tissues. Hypoxia is one of the common occurrence in injured tissues, during which angiogenesis is enhanced to improve the oxygen supply. In the present study, miR-590-3p, an anti-angiogenic miRNA, was predicted to target VEGFA, a key factor that can be transcriptionally upregulated by HIF1A during ADSC proliferation and tubule formation in response to hypoxic stimulation. Herein, we found that in response to hypoxic stimuli, HIF1A and VEGFA protein expressions were remarkably induced. In addition, ADSC viability was promoted. Incubation with conditioned medium from ADSCs stimulated by hypoxia significantly enhanced the angiogenic ability of human dermal microvascular endothelial cells (HDMECs), while the conditioned medium from VEGFA-silenced ADSCs significantly reversed the angiogenic ability of HDMECs. Regarding the molecular mechanism, it was verified that miR-590-3p binds to VEGFA; miR-590-3p inhibited VEGFA to affect the paracrine regulation by ADSCs, subsequently hindering the HDMEC angiogenesis. More importantly, the consequences of miR-590-3p-overexpressing conditioned medium on HDMEC angiogenesis were partially reversed by VEGFA-overexpressing conditioned medium. In conclusion, miR-590-3-5p/VEGFA axis modulates the paracrine secretion of VEGFA by ADSCs to affect the angiogenesis of HDMECs.

Fasentin diminishes endothelial cell proliferation, differentiation and invasion in a glucose metabolism-independent manner

The synthetic compound fasentin has been described as a modulator of GLUT-1 and GLUT-4 transporters, thus inhibiting glucose uptake in some cancer cells. Endothelial glucose metabolism has been recently connected to angiogenesis and it is now an emerging topic in scientific research. Indeed, certain compounds with a known effect on glucose metabolism have also been shown to inhibit angiogenesis. In this work we tested the capability of fasentin to modulate angiogenesis in vitro and in vivo. We show that fasentin inhibited tube formation in endothelial cells by a mechanism that involves a negative effect on endothelial cell proliferation and invasion, without affecting other steps related to the angiogenic process. However, fasentin barely decreased glucose uptake in human dermal microvascular endothelial cells and the GLUT-1 inhibitor STF-31 failed to inhibit tube formation in these cells. Therefore, this modulatory capacity on endothelial cells function exerted by fasentin is most likely independent of a modulation of glucose metabolism. Taken together, our results show a novel biological activity of fasentin, which could be evaluated for its utility in cancer and other angiogenesis-dependent diseases.

ADAM10 and Src‐signalling contribute to the inflammation‐induced formation of VE‐cadherin fragments in endothelial cells

It has been shown that the release of Vascular Endothelial‐cadherin fragments (sVE‐cadherin) is closely associated with a loss of endothelial barrier function in an acute inflammation. In this context, it has been suggested that sVE‐cadherin could be a suitable marker for early detection of systemic inflammation and sepsis. However, the detailed mechanisms underlying sVE‐cadherin formation in endothelial cells remain largely unclear. Based on previous findings that suggested a critical role for the activation of the sheddase ADAM10 (a disintegrin and metalloproteinase domain‐containing protein 10), we hypothesize that ADAM10 activation might be dependent on intracellular signaling following pro‐inflammatory stimuli.Human dermal microvascular endothelial cells (HDMECs) were incubated with pro‐inflammatory cytokine Tumor necrosis factor α (TNFα), bacterial Lipopolysaccharide (LPS) from E. coli or a combination of both to mimic acute inflammation. As revealed by ELISA‐based measurements, this resulted in increased levels of sVE‐cadherin in cell culture supernatants compared to the baseline and to untreated controls. Co‐incubation of endothelial cells with the ADAM10‐inhibitor, GI254023X, together with LPS, TNFα or both significantly attenuated inflammation‐induced increase of sVE‐cadherin levels in endothelial cell culture supernatants. Measurements of Transepithelial Electrical Resistance (TER) showed a significant loss of endothelial barrier function after application of TNFα, LPS or both compared to untreated controls. Incubation of endothelial cells with the ADAM10‐inhibitor, GI254023X, together with LPS, TNFα or both significantly blocked inflammation‐induced loss of TER. In line with this inflammation‐induced intercellular gap formation, loss of VE‐cadherin at endothelial cell borders and stress fiber formation in the actin cytoskeleton were attenuated by treatment with the ADAM10‐inhibitor. As has been described previously application of TNFα, LPS or both resulted in augmented phosphorylation of tyrosinkinase src. Importantly, using cell viability assays inflammation‐induced loss of endothelial barrier function was not caused by increased cell death under the conditions used here. The selective Src inhibitor, PP2 attenuated inflammation‐induced loss of endothelial barrier function in TER measurements. Moreover, application of PP2 blocked sVE‐cadherin release, indicating a crosstalk between inflammation‐induced activation of src and ADAM10 activation.In summary, our present data confirm previous findings demonstrating that the inhibition of ADAM10 attenuates sVE‐cadherin formation after the cells were exposed to inflammatory stimuli. Furthermore, inflammation‐induced activation of src contributes to sVE‐cadherin release which suggests that ADAM10 may be regulated by src‐dependent activation in inflammation.Support or Funding InformationYalda Ghoreishi, Sven Flemming, Nicolas SchlegelDepartment of General, Visceral, Transplant, Vascular and Pediatric Surgery University Hospital Wuerzburg, Oberduerrbacherstrasse 6, 97080 Wuerzburg, Germany

Role of WNK1/OSR1 in maintaining endothelial tight junction integrity

Deletion of the WNK1 (with no lysine kinase‐1) gene in mice causes embryonic lethality due to failed vascular development. This phenotype can be rescued by endothelial‐specific expression of WNK1 or a constitutively active form of its substrate OSR1 (Oxidative Stress Responsive‐1). Additionally, depletion of WNK1/OSR1 decreased in vitro angiogenesis and cell migration in endothelial cells. This indicates an essential function of WNK1/OSR1 in angiogenesis. However, the underlying mechanism by which WNK1/OSR1 mediates this process is unclear. WNK1 is known to regulate TGF‐β/Smad signaling which induces epithelial‐mesenchymal transition (EMT), a process utilized in cell migration and angiogenic sprouting during physiological and pathophysiological vascular remodeling. OSR1 is reported to form a complex with the TGF‐βII receptor at tight junctions which mediates dissolution of tight junction upon activation by TGF‐β. Therefore, we hypothesize that WNK1/OSR1 axis is necessary for TGF‐β/Smad‐induced disruption of cell‐cell adhesion, thereby promoting cell migration and angiogenesis. We found that WNK1 pharmacological inhibition decreased the expression of TGF‐β‐induced EMT mediators such as the receptor tyrosine kinase Axl which is known to be important for maintaining tight junction integrity in HDMECs (Human Dermal Microvascular Endothelial Cells) (Normalized densitometry values; WNKi‐ Axl: 0.2 ± 0.05 vs. siControl: 1, n=3) and in primary HUVECs (Human Umbilical Vein Endothelial Cells) (normalized densitometry values; WNK1i: 0.4 ± 0.1 vs. DMSO: 1, n=3, *p<0.05). We also determined that OSR1 and Occludin, a tight junction protein are co‐localized and that inhibition of WNK1 using a small molecule WNK1 inhibitor, decreased their co‐localization and tight junction breakdown during TGF‐β signaling in primary HUVECs (Pearson’s coefficient; Control; 0.08 ± 0.2 vs. TGF‐β: 0.61 ± 0.15 vs. TGF‐β+WNK1i: 0.12 ± 0.25, n=2, 2 replicates per experiment). Finally, we observed that bands corresponding to phosphorylated form of Occludin on a Western blot were enhanced in WNK inhibited samples compared to control (normalized densitometry values; TGF‐β+WNKi: 1 vs. TGF‐β: 0.21 ± 0.07, n=2). Since Occludin is known to be dephosphorylated during tight junction breakdown, the presence of the phosphorylated form of Occludin with WNK inhibition may indicate the disruption of cellular processes leading to TGF‐β‐dependent tight junction breakdown. These data suggest WNK1‐dependent function of OSR1 in cell migration and angiogenesis through regulating tight junction integrity. Combined, these data indicate the importance of WNK1 function in endothelial cell migration and angiogenesis. Understanding how WNK1 regulates angiogenesis and cell migration is immediately relevant during development, fibrosis as well as cancer.Support or Funding InformationAmerican Heart Association postdoctoral fellowship to Ankita Jaykumar and NIH funding R01 HL147661 to Melanie H. Cobb

THE ROLE OF THE ARP2/3 INHIBITORY PROTEIN ARPIN IN THE REGULATION OF ENDOTHELIAL BARRIER FUNCTIONS

The endothelium forms a semi‐permeable barrier in the vasculature that separates blood from underlying tissues. During inflammation, this barrier is altered usually leading to increased permeability and leukocyte recruitment. Such alterations include endothelial actin remodeling regulated by actin‐binding proteins (ABP) and junction architecture modulation. An ABP critical for actin remodeling is the Arp2/3 complex, which induces formation of branched actin networks. The Arp2/3 complex needs to be tightly regulated which is achieved by activation via nuclear promoting factors (NPF) and inhibition via proteins that locally antagonize the activity of NPFs such as PICK1, gadkin and arpin. Arpin contains a COOH‐terminal acidic motif that binds directly to the Arp2/3 complex in competition with NPF to inhibit its activity. Arpin has been shown to localize at the lamellipodial edge, where it colocalizes with the WAVE2 complex and antagonizes WAVE2 to regulate Arp2/3 activity in this specific subcellular location. However, nothing is known about the role of arpin in endothelial barrier regulation.First, we analyzed expression of arpin in different endothelial cell types and found that it is expressed at high levels in Human vein endothelial cells (HUVEC), human dermal microvascular endothelial cells (HMEC‐1), brain‐derived Endothelial cells.3 (bEnd3) and primary murine lung endothelial cells (MLEC). To analyze arpin functions in endothelial barrier regulation, we generated arpin‐depleted HUVEC by transfecting them with lentiviruses encoding specific short hairpin RNA (shRNA) against arpin and a scrambled control sequence, and selecting stable clones using puromycin. Western blot analysis revealed arpin downregulation of more than 90%. Expression of the junctional proteins VE‐cadherin, claudin‐1, occludin, ZO‐1 and the apical adhesion molecule ICAM‐1 did not change in arpin‐depleted HUVECs. However, VE‐Cadherin staining revealed significant gaps at cell contacts and redistribution into the cytoplasm. Moreover, we consistently observed more actin filaments in arpin‐depleted HUVEC. As ROCK1 and ZIPK kinases were overexpressed in the absence of arpin, we conclude that these filaments are contractile stress fibers that destabilize junctions by generating pulling forces. In vitro filter‐based permeability assays showed that arpin‐depleted HUVEC are much more permeable compared to control cells transduced with lentivirus expressing scrambled shRNA. These data suggest that arpin regulates the endothelial barrier by two possible mechanisms: First, arpin may inhibit Arp2/3 to control junction protein internalization and vesicle trafficking; and second, arpin may inhibit Arp2/3 to control actin dynamics at junctions. Whether arpin additionally regulates Arp2/3‐independent processes is currently unknown, and will be investigated in the future. Our data reveal arpin as a new critical regulator of endothelial barrier integrity.Support or Funding InformationThis work was supported by a grant of the Mexican Council for Science and Technology (CONACyT, 284292 to MS).

A Novel Model of Long‐term Human Microvascular Endothelial Cell Serum Starvation

Microvascular endothelial cell injury and progressive loss of microvasculature in the dermis, kidney, retina and perineural beds is caused by hyper‐and hypoglycemic insults in individuals with Diabetes mellitus (DM). The consequence of microvascular injuries occurring over years to decades is the onset of DM‐associated co‐morbidities such as delayed wound healing, renal failure, vision loss and peripheral neuropathy. The Diabetes Control and Complications Trial noted a correlation between baseline endogenous C‐peptide serum levels and the delay in onset of DM‐associated co‐morbidities. In immortalized human microvascular endothelial cells (HMEC) and primary human dermal microvascular endothelial cells treatment with 1.5nM C‐peptide and 0.5nM insulin prevented hyperglycemia‐induced oxidative injury. In studies of these cells using immunofluorescence to track C‐peptide and the C‐peptide receptor, G‐protein coupled receptor 146 (GPR146), endothelial cells repeatedly demonstrated cytosolic stores of C‐peptide. Fetal bovine serum, a component of almost all cell culture growth media was determined to have high concentrations of C‐peptide. The aim of this study was to develop a model of long term endothelial serum starvation that would result in depleted internal stores of C‐peptide, but would prevent endothelial cells transformation to smooth muscle cells or fibroblasts, as endothelial progenitor cells are capable of differentiating into one of these three cell types. We hypothesized that cytosolic C‐peptide would reduce over time with serum starvation. To test this hypothesis, HMEC were incubated for up to two months in MCDB13 media supplemented with human epidermal growth factor, hydrocortisone, vascular endothelial growth factor, basic fibroblast growth factor, insulin‐like growth factor‐1, ascorbic acid, and L‐glutamine. Serum starved HMEC were harvested at various time points and stained for C‐peptide (MAB80561 R&D Systems clone 915612) and DAPI or lysed with lysis buffer with 2‐mercaptoethanol followed by RNA isolation, cDNA preparation and RT‐PCR evaluation of targets. Cytosolic C‐peptide fluorescent signal diminished over time in a linear manner but was never completely abolished. HMEC serum starved for 30 days retained endothelial markers CD31, CD34, VEGFR2, had no change in smooth muscle markers Smoothelin, or Smooth muscle actin, and similarly, had no change in expression Vimentin a fibroblast marker relative to un‐serum starved HMEC controls as determined by RT‐PCR. The ability to serum starve HMEC long‐term and substantially reduce internal C‐peptide stores has the potential to enhance research to elucidate the pathways employed by C‐peptide in the prevention of hyperglycemia‐induced microvascular injury.Support or Funding InformationNIDDK

Neutrophil-derived exosome from systemic sclerosis inhibits the proliferation and migration of endothelial cells

ObjectivesSystemic sclerosis (SSc) is an autoimmune disease characterized by vasculopathy, inflammation, and extensive fibrosis in multiple organs. Exosomes (EXOs) are cell-derived vesicles contained various DNAs, RNAs and proteins, and play important roles in various diseases. Here, we aimed to investigate the roles of SSc EXOs in angiogenesis related mechanisms. MethodsEXOs were isolated from plasma, cultured peripheral blood mononuclear cells (PBMCs)/neutrophil supernatants, and identified by transmission electron microscopy. The expression of S100A8/A9 was measured by real-time PCR and ELISA. Proliferation, migration and scratch assays in human dermal microvascular endothelial cells (HDMECs) were used to study the EXOs influence. ResultsPlasma and neutrophil EXOs from SSc patients can suppress the proliferation and migration of HDMECs. High levels of S100A8/A9 were found in SSc EXOs which derived from plasma, PBMCs and neutrophils. The expression of S100A8/A9 in neutrophil EXOs was higher than that in PBMC EXOs in SSc patients. The proliferation and migration of HDMECs were possibly inhibited by S100A8/A9 of neutrophil EXOs. ConclusionsNeutrophil EXOs from SSc patients inhibits the proliferation and migration of HDMECs, S100A8/A9 might play an important role in this process.

Advanced gelatin-based vascularization bioinks for extrusion-based bioprinting of vascularized bone equivalents

Bone tissue is highly vascularized. The crosstalk of vascular and osteogenic cells is not only responsible for the formation of the strongly divergent tissue types but also for their physiological maintenance and repair. Extrusion-based bioprinting presents a promising fabrication method for bone replacement. It allows for the production of large-volume constructs, which can be tailored to individual tissue defect geometries. In this study, we used the all-gelatin-based toolbox of methacryl-modified gelatin (GM), non-modified gelatin (G) and acetylated GM (GMA) to tailor both the properties of the bioink towards improved printability, and the properties of the crosslinked hydrogel towards enhanced support of vascular network formation by simple blending. The vasculogenic behavior of human dermal microvascular endothelial cells (HDMECs) and human adipose-derived stem cells (ASCs) was evaluated in the different hydrogel formulations for 14 days. Co-culture constructs including a vascular component and an osteogenic component (i.e. a bone bioink based on GM, hydroxyapatite and ASCs) were fabricated via extrusion-based bioprinting. Bioprinted co-culture constructs exhibited functional tissue-specific cells whose interplay positively affected the formation and maintenance of vascular-like structures. The setup further enabled the deposition of bone matrix associated proteins like collagen type I, fibronectin and alkaline phosphatase within the 30-day culture.

Purpuric drug eruptions induced by EGFR tyrosine kinase inhibitors are associated with IQGAP1-mediated increase in vascular permeability.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are used as a treatment for non-small-cell lung cancer. There have been some reports of EGFR-TKIs being associated with vascular adverse events. We found that EGFR-TKIs decreased the proliferation of HMEC-1s (immortalized human dermal microvascular endothelial cells) and HMVECs (human dermal microvascular endothelial cells), and also inhibited the phosphorylation of EGFR and ERK. We examined the mRNA expression profile of erlotinib-treated HMEC-1s and identified IQ motif containing GTPase activating protein 1 (IQGAP1) as the most consistently up-regulated transcript and protein. IQGAP1 was also overexpressed and co-localized with endothelial cells in the lesional skin. Notably, increased IQGAP1 expression was associated with decreased transendothelial electrical resistance and increased vascular permeability in vitro. Erlotinib treatment enriched the staining of IQGAP1 and reduced the intensities of α-catenin at the sites of cell-cell contact. In conclusion, erlotinib induces adherens junction dysfunction by modulating the expression of IQGAP1 in dermal endothelial cells. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The profiles of miRNAs and lncRNAs in peripheral blood neutrophils exosomes of diffuse cutaneous systemic sclerosis

BackgroundDiffuse cutaneous systemic sclerosis (dSSc) is a systemic autoimmune disease with skin fibrosis. Neutrophils display important roles in autoimmunity, inflammation, vasculopathy and fibrosis. Exosomes (EXOs) are cell-derived vesicles contained various noncoding RNAs, mRNA and proteins with biological roles. ObjectiveTo investigate the roles of miRNAs and lncRNAs from dSSc neutrophils EXOs. MethodsEXOs were isolated from cultured neutrophils supernatants and identified by transmission electron microscopy. Global expression of miRNAs and lncRNAs in neutrophils EXOs were sequenced by Illumina HiSeq 3000 and bioinformatic analyses were performed by R/Bioconductor. Genes were validated by real-time quantitative PCR. ResultsIn profiles of neutrophils EXOs, we identified 22 dysregulated miRNAs and 281 dysregulated lncRNAs. Predicted target genes of them were enriched in GO, KEGG and Reactome pathways, Wnt, AMPK, IL-23 and NOTCH signaling pathways were selected for further analysis. Widely interactions among them were also found. Human dermal microvascular endothelial cells and human primary skin fibroblasts were stimulated with dSSc neutrophils EXOs, these fibrosis related genes were detected and some changes were found, such as ENST00000533886.1-hsa-miR-1268a-CAMK2G in Wnt and IL-23 signaling pathways, ENST00000610091.1-hsa-miR-299-3p, 512-3p-CPT1A in IL-23 and AMPK signaling pathways, NR_001564.2, ENST00000520562.1, ENST00000596567.1-hsa-miR-299-3p, 512-3p -TFDP2 in IL-23, AMPK and NOTCH signaling pathways. ConclusionsThe profiles of miRNAs and lncRNAs of neutrophils EXOs provided novel clues for dSSc pathogenesis. We identified several gene pairs in the Wnt, AMPK, IL-23 and NOTCH signaling pathways, which could be potential biomarkers and therapeutic targets in dSSc.

Telocytes inhibited inflammatory factor expression and enhanced cell migration in LPS-induced skin wound healing models in vitro and in vivo

BackgroundCell proliferation and death are key components of wound healing and tissue repair. Telocytes (TCs) represent a newly discovered cell type that can protect tissue from acute injury via cell–cell communication with adjacent cells. The aim of this study was to use a mouse model of skin wound healing and lipopolysaccharide (LPS)-induced cell injury to evaluate the effects of TCs on skin wound healing in vivo and in vitro.Material/methodsImmunohistochemical staining was performed to evaluate the alteration of TCs in tissues from normal and chronic wound patients. Then, a male C57BL/6 mouse wound model of the back was established. The mice were divided randomly into three groups, and wound healing was estimated according to the wound healing rate and histology. An LPS-induced co-culture model of a mouse lung telocyte cell line (TCs) with human keratinocyte (HaCaT), human dermal microvascular endothelial cell (HDMEC) or murine fibroblast (L929) cell lines was established to analyse the effects of TCs on constitutive cell types of the skin. Cell proliferation, migration and apoptosis were examined, and reactive oxygen species (ROS) and inflammatory factors in HaCaT cells, HDMECs, and L929 cells were detected to study the mechanisms involved in TC protection in skin wounds.ResultsTCs were significantly increased in tissues from chronic wound patients compared with healthy controls. Wound healing was significantly improved in wound mouse models treated with exogenous TCs compared with LPS-induced models. TCs reversed the LPS-induced inhibition of HaCaT cells and HDMECs and reduced the LPS-induced apoptosis of HaCaT cells and the death ratios of HDMECs and L929 cells. TCs reversed LPS-induced ROS in HDMECs and L929 cells and decreased inflammatory factor mRNA levels in HaCaT cells, HDMECs and L929 cells.ConclusionsTCs reduce wound healing delay, and inflammatory responses caused by LPS might be mediated by inflammatory inhibition, thus restricting apoptosis and promoting migration of the main component cell types in the skin.

Deficiency of \u03b1II-spectrin affects endothelial cell\u2013matrix contact and migration leading to impairment of angiogenesis in vitro

BackgroundPrecise coordination of cytoskeletal components and dynamic control of cell adhesion and migration are required for crucial cell processes such as differentiation and morphogenesis. We investigated the potential involvement of αII-spectrin, a ubiquitous scaffolding element of the membrane skeleton, in the adhesion and angiogenesis mechanism.MethodsThe cell models were primary human umbilical vein endothelial cells (HUVECs) and a human dermal microvascular endothelial cell line (HMEC-1). After siRNA- and shRNA-mediated knockdown of αII-spectrin, we assessed its expression and that of its partners and adhesion proteins using western blotting. The phenotypes of the control and spectrin-depleted cells were examined using immunofluorescence and video microscopy. Capillary tube formation was assessed using the thick gel Matrigel matrix-based method and a microscope equipped with a thermostatic chamber and a Nikon Biostation System camera.ResultsKnockdown of αII-spectrin leads to: modified cell shape; actin cytoskeleton organization with the presence of peripheral actin patches; and decreased formation of stress fibers. Spectrin deficiency affects cell adhesion on laminin and fibronectin and cell motility. This included modification of the localization of adhesion molecules, such as αVβ3- and α5-integrins, and organization of adhesion structures, such as focal points. Deficiency of αII-spectrin can also affect the complex mechanism of in vitro capillary tube formation, as demonstrated in a model of angiogenesis. Live imaging revealed that impairment of capillary tube assembly was mainly associated with a significant decrease in cell projection length and stability. αII-spectrin depletion is also associated with significantly decreased expression of three proteins involved in capillary tube formation and assembly: VE-cadherin, MCAM and β3-integrin.ConclusionOur data confirm the role of αII-spectrin in the control of cell adhesion and spreading. Moreover, our findings further support the participation of αII-spectrin in capillary tube formation in vitro through control of adhesion molecules, such as integrins. This indicates a new function of αII-spectrin in angiogenesis.

Pilot Study of the Biological Properties and Vascularization of 3D Printed Bilayer Skin Grafts.

The skin is the largest human organ, and defects in the skin with a diameter greater than 4 cm do not heal without treatment. Allogeneic skin transplantation has been used to allow wound healing, but many grafts do not survive after implantation, due to multiple complications in the procedure. In the present study, the vascularization of three-dimensional (3D) printed full-thickness skin grafts was investigated. Dermal-epithelial grafts were transplanted into a nude mouse model to evaluate integration with the host tissue and the extent of wound healing. To create microvessels in the skin grafts, a bilayer structure consisting of human dermal fibroblasts, keratinocytes, and microvascular endothelial cells was designed and fabricated using an extruded 3D printer. Human dermal fibroblasts and human microvascular endothelial cells were mixed with gelatin-sodium alginate composite hydrogel as the dermis, and human keratinocytes were mixed with gel as the epithelium. Confocal imaging allowed visualization of the location of the cells in the double-layer skin grafts. A full-thickness wound was created on the backs of nude mice and then covered with a double-layer skin graft. Various groups of mice were tested. Animals were euthanized and tissue samples collected after specified time points. Compared with the control group, wound contraction improved by approximately 10%. Histological analysis demonstrated that the new skin had an appearance similar to that of normal skin and with a significant degree of angiogenesis. The results of the immunohistochemical analysis demonstrated that the transplanted cells survived and participated in the healing process.

Porphyromonas gingivalis W83 traffics via ICAM1 in microvascular endothelial cells and alters capillary organization in vivo

ABSTRACT Objective: Microvascular dysfunction is a feature of periodontal disease. P. gingivalis, one of the most common oral bacteria present in gingival tissue biofilms, has also been identified in the gingival capillaries of patients with chronic periodontitis. We sought to determine the effect of P. gingivalis W83 infection on microvascular endothelium in vivo and in vitro. Methods and Results: Interdental papillae of rats with P. gingivalis-induced alveolar bone loss had a more dilated and denser subepithelial capillary network than uninfected controls. P. gingivalis W83 was detected in the epithelial layers, the subepithelial connective tissue matrix, and subgingival capillaries. P. gingivalis invaded human dermal microvascular endothelial cells (HD-MVECS) and persisted up termination (24 h). Colocalization analysis at 2.5, 6, and 24 h post-inoculation showed that 79–88% of internalized bacteria were in ICAM-1 positive endosomes, and 10–39% were in Rab5, Rab7, or LAMP1 positive compartments, but never in autophagosomes. Antibody-based blockade of ICAM-1 significantly reduced W83 invasion in HD-MVECS. P. gingivalis infected HD-MVECS were unable to form vascular networks in Matrigel. Conclusions: P. gingivalis perturbs microvascular endothelial function and invasion of these cells via ICAM-1 may be important for microbial persistence within tissues.

Ciclopirox olamine promotes the angiogenic response of endothelial cells and mesenchymal stem cells.

Prolyl hydroxylase inhibitors (PHIs) are promising compounds to promote angiogenesis by stabilizing hypoxia-inducible factor-1α (HIF-1α), a master regulator of angiogenesis. Increased HIF-1α presence induces expression of proangiogenic genes such as vascular endothelial growth factor (VEGF). We investigated the pharmacological induction of hypoxia via the PHI ciclopirox olamine (CPX) as angiogenesis strategy on human dermal microvascular endothelial cell (hd-mvEC) spheroids directly and indirectly via activating human mesenchymal stem cells (hMSCs). HMSCs were isolated from bone marrow and hd-mvECs from foreskin biopsies. MSC-conditioned medium after CPX stimulation (MSC-CM CPX) was analyzed by VEGF ELISA and Proteome Profiler™ Human Angiogenesis Array. Direct stimulation with CPX and indirect stimulation via MSC-CM CPX were compared in sprouting assays of hd-mvEC spheroids. Direct stimulation with CPX significantly increased sprouting of hd-mvEC spheroids. MSC-CM CPX also induced sprouting from hd-mvEC spheroids, which was mediated by angiogenic VEGF and other proangiogenic factors that had been produced by stimulated hMSCs. The stimulation with CPX increased the proangiogenic response of hd-mvECs and hMSCs. The direct stimulation of hd-mvECs with CPX has the potential to replace external VEGF supplementation. Thus, CPX can induce angiogenesis in ECs even in the absence of auxiliary cells demonstrating a promising proangiogenic approach.

Fli1 deficiency induces endothelial adipsin expression, contributing to the onset of pulmonary arterial hypertension in systemic sclerosis.

Adipsin, or complement factor D, is a serine proteinase catalysing complement factor C3 breakdown, leading to the production of opsonin (C3b), membrane attack complex (C5b-C9) and anaphylatoxins (C3a and C5a). Since adipsin is potentially associated with pulmonary arterial hypertension in SSc, we investigated adipsin expression in dermal small vessels of SSc-involved skin, the mechanism regulating adipsin expression in endothelial cells, and the correlation of serum adipsin levels with SSc clinical symptoms. Adipsin expression was assessed by immunohistochemistry with skin sections of SSc and healthy subjects. mRNA levels of target genes and transcription factor binding to the ADIPSIN promoter were evaluated by quantitative reverse transcription PCR and chromatin immunoprecipitation, respectively. Serum adipsin levels were determined by enzyme-linked immunosorbent assay. Adipsin expression was remarkably increased in dermal small vessels of SSc-involved skin as compared with those of healthy control skin. Consistent with the notion that Fli1 deficiency induces SSc-like phenotypes in various types of cells, FLI1 siRNA enhanced adipsin expression at protein and mRNA levels and Fli1 bound to the ADIPSIN promoter in human dermal microvascular endothelial cells. Serum adipsin levels were significantly lower in diffuse cutaneous SSc patients than in limited cutaneous SSc patients and healthy controls, and were associated positively with elevated right ventricular systolic pressure and inversely with interstitial lung disease by multivariate regression analysis. Adipsin is up-regulated at least partially by Fli1 deficiency in endothelial cells, potentially contributing to the development of pulmonary vascular involvement in SSc.

Fibrin \u03b3/\u03b3' influences the secretion of fibrinolytic components and clot structure

BackgroundIn healthy subjects fibrinogen γ/γ‘ circulates at 8–15% of the total plasma fibrinogen concentration. Elevated levels of this variant have been associated with arterial thrombosis, and its diminution with venous thrombosis. The aims of the present work were to analyze the structure of the fibrin network formed on the top of human dermal microvascular endothelial cells (HMEC-1) at different fibrinogen γ/γ‘ concentrations, as well as its influence on the secretion of fibrinolytic components.The kinetics of fibrin polymerization on top of HMEC-1 cells with 3, 10, and 30% fibrinogen γ/γ‘ was followed at 350 nm. The secretion of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI 1) by HMEC-1 were measured in the supernatant and cell lysates, after incubation with 1 nM thrombin, fibrin with 3, and 30% fibrinogen γ/γ‘, using commercial kits. The influence of fibrinogen γ/γ‘ on fibrin structure on the surface of the HMEC-1 was followed with laser scanning confocal microscopy (LSCM).ResultsThe kinetics of fibrin formation on HMEC-1 with 3 and 10% fibrinogen γ/γ‘ were similar. However, with 30% fibrinogen γ/γ‘ both the slope and final turbity were approximately 50% less. The LSCM images showed the dramatic effects of increasing fibrinogen γ/γ‘ from 3 to 30%. The uPA and PAI 1 concentrations in culture supernatants HMEC-1 cells treated with thrombin or 30% γ/γ‘ fibrin were two-fold increased as compared to basal culture supernatants and 3% γ/γ‘ fibrin-treated HMEC-1. In all stimulatory conditions the intracellular concentration of uPA was higher than in supernatants. In contrast, the intracellular PAI 1 concentration was decreased as compared to that measured in the supernatant, including the basal condition.ConclusionA concentration of 30% fibrin γ/γ‘ alter drastically fibrin structure on the cell surface and affects the secretion of uPA and PAI 1 through its capacity to bind thrombin.

Triptolide inhibits angiogenesis in microvascular endothelial cells through regulation of miR-92a.

Atherosclerosis is one common chronic inflammatory disease in which angiogenesis is involved. Here we established an in vitro cell model of angiogenesis made by human dermal microvascular endothelial cells (HMEC-1) and work to investigate the role of triptolide (TPL) in this model. To induce angiogenesis, HMEC-1 cells were cultured in Matrigel-conditioned medium. The ratio of tubes to nucleus was detected. To evaluate angiogenesis, Western blot assay was carried out to detect endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor receptor-2 (VEGFR2) and VEGF. Cell counting kit-8 was utilized to estimate the viability of HMEC-1 cells. microRNA (miR)-92a was analyzed by qRT-PCR. The targeting relationship between integrin subunit alpha 5 (ITGA5) and miR-92a was verified through luciferase activity assay. The effects of ITGA5 on signaling transducers (ERK, PI3K, and AKT) in a phosphorylated form were valued using Western blot method. After stimulated by TPL, LY294002 and PD98059, the alteration in phosphorylation of the signaling transducers was evaluated by Western blot assay. The ratio of tubes to nucleus and angiogenesis related factors were increased with the delaying of culture time. TPL decreased the expression of angiogenesis factors. Furthermore, miR-92a was upregulated by TPL and miR-92a silence upregulated angiogenesis factors. In addition, TPL decreased ITGA5 which was proved as a target of miR-92a. ITGA5 overexpression resulted in the abundance of angiogenesis factors while ITGA5 silence led to the opposite results. Meanwhile, ITGA5 overexpression increased phosphorylation of ERK, PI3K and AKT while ITGA5 silence reversed the trend. TPL (as an anti-angiogenesis agent) suppressed angiogenesis by upregulating miR-92a, and miR-92a-mediated down-regulation of ITGA5 blocked the signaling transduction of ERK and PI3K/AKT pathways.

Royal jelly regulates the proliferation of human dermal microvascular endothelial cells through the down-regulation of a photoaging-related microRNA.

Although royal jelly is believed to prevent skin aging, the underlying mechanism is not known in detail. In the present study, we investigated the plausibility of the involvement of microRNAs in the manifestation of this effect of royal jelly. The expression of microRNAs was determined by PCR array analysis and real-time PCR and the number of cells was counted with a cell counter. Using PCR array, we identified four microRNAs that were downregulated by royal jelly in cultured human dermal microvascular endothelial cells (HDMEC). Upon comparison of the expression of the four microRNAs between young and senescent facial skin, miR-129-5p was found to be significantly upregulated in senescent skin. Consistently, the expression of miR-129-5p in HDMEC was significantly increased by UVB radiation, suggesting that this microRNA is related to photoaging. The royal jelly treatment increased the number of HDMEC. Furthermore, forced overexpression of miR-129-5p resulted in significant decrease in the number of HDMEC, and its forced downregulation increased the number of cells. The number and density of vessels is reported to be decreased in aged skin. Our results indicate that miR-129-5p is induced in damaged endothelial cells upon exposure to UV radiation, which decreases the cell number. Furthermore, administration of royal jelly downregulated the expression of miR-129-5p in endothelial cells, and might prevent skin aging by maintaining the number of cells. The present study elucidates the mechanism of vessel aging caused by UV exposure and the anti-aging effects of royal jelly through the involvement of microRNA.

HIF1α/miR-199a/ADM feedback loop modulates the proliferation of human dermal microvascular endothelial cells (HDMECs) under hypoxic condition

ABSTRACT Hypoxia-inducible factor 1α (HIF1α) plays a protective role in the hypoxia-induced cellular injury. In the present study, we attempted to investigate the role and mechanism of HIF1αin human dermal microvascular endothelial cells (hDMECs), a common-used cell model for researches on the hypoxia-induced injury during skin wounds healing. As revealed by ChIP and online tools prediction and confirmed by luciferase reporter and ChIP assays, HIF1A can bind to the promoter regions of ADM and miR-199a, while miR-199a directly binds to the 3ʹUTR of HIF1A and ADM. Hypoxia stress induces HIF1α and ADM expression while inhibits miR-199a expression. Under hypoxic condition, HIF1α knockdown increases the nucleus translocation of p65 and the release of TNF-α and IL-8, inhibits the proliferation and migration, while promotes the cellular permeability in HDMECs upon hypoxic stress, while ADM overexpression and miR-199a inhibition exerted an opposite effect on HDMECs. ADM overexpression or miR-199a inhibition could partially reverse the effect of HIF1A knockdown under hypoxia. In summary, we demonstrate a feedback loop consists of HIF1α, miR-199a, and ADM which protect HDMECs from hypoxia-induced cellular injury by modulating the inflammation response, cell proliferation, migration and permeability in HDMECs.