12/15-Lipoxygenase gene knockout severely impairs ischemia-induced angiogenesis due to lack of Rac1 farnesylation

Previously, we have reported that 15(S)‐hydroxyeicosatetraenoic acid (15(S)‐HETE)‐induced migration and tube formation of endothelial cells require Rac1 activation. To understand the mechanisms by which 15(S)‐HETE activates Rac1, here, we have studied the role of HMG‐CoA reductase and RhoGFP, α‐Pix. 15(S)‐HETE by inducing HMG‐CoA reductase expression caused increased farnesylation and membrane translocation of Rac1, where it became activated by Src‐dependent α‐Pix stimulation in human dermal microvascular endothelial cells (HDMVECs) and these events enhanced the migration and tube formation of these cells. Mevalonate rescued 15(S)‐HETE‐induced Rac1 farnesylation in HDMVECs and the migration and tube formation of these cells from inhibition by simvastatin. Hind limb ischemia induced Rac1 farnesylation and activation leading to increased angiogenesis and these effects were blocked by simvastatin and rescued by mevalonate in C57BL/6 mice. In contrast, hind limb ischemia failed to induce Rac1 farnesylation and activation as well as angiogenic response in 12/15‐Lox−/− mice. Activation of Src and α‐Pix were also compromised at least to some extent in 12/15‐Lox−/− mice. Together, these findings demonstrate that HMG‐CoA reductase‐dependent farnesylation and α‐Pix‐dependent GDP/GTP exchange of Rac1 is essential for 12/15‐Lox‐12/15 (S)‐HETE‐induced angiogenesis.

To understand the involvement of matrix metalloproteinases (MMPs) in 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE)-induced angiogenesis, we have studied the role of MMP-2. 15(S)-HETE induced MMP-2 expression and activity in a time-dependent manner in human dermal microvascular endothelial cells (HDMVECs). Inhibition of MMP-2 activity or depletion of its levels attenuated 15(S)-HETE-induced HDMVEC migration, tube formation, and Matrigel plug angiogenesis. 15(S)-HETE also induced Fra-1 and c-Jun expression in a Rac1-MEK1-JNK1-dependent manner. In addition, 15(S)-HETE-induced MMP-2 expression and activity were mediated by Rac1-MEK1-JNK1-dependent activation of AP-1 (Fra-1/c-Jun). Cloning and site-directed mutagenesis of MMP-2 promoter revealed that AP-1 site proximal to the transcriptional start site is required for 15(S)-HETE-induced MMP-2 expression, and Fra-1 and c-Jun are the essential components of AP-1 that bind to MMP-2 promoter in response to 15(S)-HETE. Hind limb ischemia led to an increase in MEK1 and JNK1 activation and Fra-1, c-Jun, and MMP-2 expression resulting in enhanced neovascularization and recovery of blood perfusion in wild-type mice as compared with 12/15-Lox(-/-) mice. Together, these results provide the first direct evidence for a role of 12/15-Lox-12/15(S)-HETE axis in the regulation of ischemia-induced angiogenesis.

To determine the efficacy of cytochrome P450 2C9 metabolites of arachidonic acid, viz. 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs), in inducing angiogenesis, we have studied their effects on human dermal microvascular endothelial cell (HDMVEC) tube formation and migration. All four EETs stimulated HDMVEC tube formation and migration in a dose-dependent manner. Because 14,15-EET was found to be slightly more efficacious than 5,6-, 8,9-, and 11,12-EETs in stimulating HDMVEC tube formation and migration, we next focused on elucidation of the signaling mechanisms underlying its angiogenic activity. 14,15-EET stimulated Akt and S6K1 phosphorylation in Src- and phosphatidylinositol 3-kinase (PI3K)-dependent manner in HDMVECs. Inhibition of Src and PI3K-Akt-mTOR signaling by both pharmacological and dominant-negative mutant approaches suppressed 14,15-EET-induced HDMVEC tube formation and migration in vitro and Matrigel plug angiogenesis in vivo. In addition, 14,15-EET induced the expression of fibroblast growth factor-2 (FGF-2) in Src- and PI3K-Akt-dependent and mTOR-independent manner in HDMVECs. Neutralizing anti-FGF-2 antibodies completely suppressed 14,15-EET-induced HDMVEC tube formation and migration in vitro and Matrigel plug angiogenesis in vivo. Together, these results show for the first time that Src and PI3K-Akt signaling via targeting in parallel with FGF-2 expression and mTOR-S6K1 activation plays an indispensable role in 14,15-EET-induced angiogenesis.

Emerging evidence supports that angiogenesis is essential for the wound healing of diabetic foot ulcer (DFU), and high glucose (HG)-induced dysfunction of human dermal microvascular endothelial cells is a key factor that hinders angiogenesis. However, the underlying mechanisms by which HG leads to the dysfunction of human dermal microvascular endothelial cells has not been fully elucidated. In the present investigation, we discovered a significant upregulation of the long non-coding RNA GFOD1-AS1(GFOD1-AS1) in the ulcer margin samples of patients with DFU and the HG-induced dysfunction model of human dermal microvascular endothelial cells, attributing its dysregulation to the stabilizing effect of NAT10-mediated ac4C modification, as corroborated by an integrated approach of data mining and experimental validation. Subsequently, a series of in vitro functional analyses showed that ectopic expression of GFOD1-AS1 promoted impaired function of human dermal microvascular endothelial cells. In contrast, knockdown of GFOD1-AS1 significantly alleviated the HG-induced functional impairment in human dermal microvascular endothelial cells, as indicated by the enhanced cell proliferation, migration, and tube formation. Mechanistically, GFOD1-AS1 directly interacts with DNA methyltransferase DNMT1 to block its ubiquitin-proteasome degradation, thereby enhancing the protein stability of DNMT1.This stability elevates DNMT1 protein expression, ultimately inducing HG-induced dysfunction in human dermal microvascular endothelial cells. In summary, our results reveal that GFOD1-AS1 serves as a potential therapeutic target for DFU, and highlight the critical role of the NAT10/GFOD1-AS1/DNMT1 axis in the dysfunction of human dermal microvascular endothelial cells in DFU.

Interaction of HSV-1 Infected Peripheral Blood Mononuclear Cells with Cultured Dermal Microvascular Endothelial Cells: a Potential Model for the Pathogenesis of HSV-1 Induced Erythema Multiforme

Expression of Proopiomelanocortin Peptides in Human Dermal Microvascular Endothelial Cells: Evidence for a Regulation by Ultraviolet Light and Interleukin-1

Arachidonic acid and its metabolites have been previously implicated in the regulation of endothelial cell proliferation. Arachidonic acid may be liberated from cellular phospholipids by the action of group VIA calcium-independent phospholipase A2 (iPLA2-VIA). Consequently, we tested the hypothesis that iPLA2-VIA activity is linked to the regulation of endothelial cell proliferation. Inhibition of iPLA2 activity by bromoenol lactone (BEL) was sufficient to entirely block endothelial cell growth. BEL dose-dependently inhibited endothelial cell DNA synthesis in a manner that was reversed upon the exogenous addition of arachidonic acid. DNA synthesis was inhibited by the S-isomer and not by the R-isomer of BEL, demonstrating that endothelial cell proliferation is mediated specifically by iPLA2-VIA. iPLA2-VIA activity was critical to the progression of endothelial cells through S phase and is required for the expression of the cyclin A/cdk2 complex. Thus, inhibition of iPLA2-VIA blocks S phase progression and results in exit from the cell cycle. Inhibition of iPLA2-VIA-mediated endothelial cell proliferation is sufficient to block angiogenic tubule formation in co-culture assays. Consequently, iPLA2-VIA is a novel regulator of endothelial cell S phase progression, cell cycle residence, and angiogenesis.

The events that lead to irreversible neutrophil-mediated cell damage are poorly understood. Evidence indicates that the mechanism of injury varies with regard to cell type and the means of neutrophil (PMN) stimulation. The molecular basis of neutrophil-mediated cell injury and inhibitors of this process were evaluated in two in vitro assays: a neutrophil-mediated cytotoxicity assay, which measures the release of endothehal bound radiolabel and detects both cell injury and detachment; and a cytochrome c reduction assay which measures neutrophil-derived superoxide (O2-) activity. Human aortic endothelial (HAE) and human dermal microvascular endothelial (HDME) cells were found to differ in susceptibility to neutrophil-mediated injury. The amount of HAE cell injury correlated well with the amount of Or generated by stimulated PMN. HDME cells were injured in the presence of lower concentrations O2-than HAE cells and by PMN stimulated with inflammatory mediators (LPS, PAF, and fMLP) which appeared to generate little or no O2-. Neutrophil-mediated injury to both HAE and HDME cells was found, however, to be inhibitable by superoxide dismutase (SOD) in a dose dependent manner. The injury was not abrogated by catalase of the iron chelator, deferoxamine mesylate (desferol). When the amount of nitric oxide (NO) released from endothelial cells and unstimulated PMN (measured as nitrite) was examined, HDME cells and PMN were found to release significantly more NO than HAE cells. Stimulation of PMN to generate O2-resulted in significant decreases in basal nitrite levels. Furthermore, induction of NO synthesis by bradykinin or addition of exogenous NO significantly enhanced neutrophilmediated HAE cell injury. This injury was abolished by SOD. These results indicate that the superoxide anion is directly involved in the neutrophil-mediated killing of HAE and HDME cells. These data also suggest a role for peroxynitrite (formed upon interaction of NO and O2-) in oxidative damage. Differential release of NO from cell types may explain variation in susceptibility to neutrophil-mediated injury.

C-C Motif Chemokine 8 promotes angiogenesis in vascular endothelial cells.

Upregulation of adhesion molecules on endothelial cells following irradiation has been shown, but the functional significance of this upregulation in various endothelial cell lines is not clear. We have developed an in vitro flow model to study the functional consequences of the radiation-induced upregulation of E-selectin and intercellular adhesion molecule (ICAM-1). Human dermal microvascular endothelial cells (HDMEC), human umbilical vein endothelial cells (HUVEC), or transformed human microvascular endothelial cells (HMEC-1) were grown in 35-mm dishes and irradiated with a single dose of 10 Gy. HL-60 (human promyelocytic leukemia) cells were perfused over the irradiated endothelial cells in a parallel plate flow chamber at shear stress ranging from 0.5 to 2.0 dynes/cm2. Flow cytometry was used to quantify the expression of E-selectin and ICAM-1 on the various endothelial cells. Flow cytomeric analysis revealed an upregulation of ICAM-1 expression on all three cell types postirradiation (post-IR), and an upregulation of E-selectin expression only on HDMEC post-IR. E-selectin expression was detected on control HDMEC, but at a lower level than that detected on post-IR HDMEC. Flow assays revealed a significant increase in the number of rolling and firmly adherent HL-60 cells on post-IR HDMEC at shear stress < or =1.5 dynes/cm2; pretreatment of control and irradiated HDMEC with antibodies to E-selectin and ICAM-1 significantly diminished the number of rolling and firmly adherent HL-60 cells, respectively. No rolling or firm adhesion of HL-60 cells was observed on HUVEC or HMEC-1 monolayers post-IR. These findings suggest that ICAM-1 is upregulated on irradiated HDMEC, HUVEC, and HMEC-1. E-selectin is upregulated to a functional level only on irradiated HDMEC, and not on irradiated HUVEC or HMEC-1.

Rheumatoid arthritis (RA) is characterized by leukocyte recruitment and angiogenesis. We investigated the effects of sulfasalazine (SSZ) and its metabolites, sulfapyridine (SP) and 5-aminosalicylic acid (5-ASA), on components of angiogenesis, namely, endothelial cell (EC) chemotaxis and proliferation, as well as on EC chemokine and soluble adhesion molecule expression. SSZ, SP, and 5-ASA were assayed for their effects on basic fibroblast growth factor (bFGF)-induced human dermal microvascular endothelial cell (HMVEC) chemotaxis and proliferation. EC were plated on Matrigel to assess the effect of SSZ on EC tube formation. Enzyme-linked immunosorbent assays were performed to determine changes in HMVEC production of interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), growth-related oncogene alpha (GROalpha), epithelial neutrophil-activating peptide 78 (ENA-78), soluble E-selectin (sE-selectin), and soluble intercellular adhesion molecule 1 (sICAM-1) upon treatment with SSZ or its metabolites. HMVEC incubated with SSZ or SP exhibited reduced bFGF-induced chemotaxis (59%, [n = 7] and 22%, [n = 3], respectively) (P<0.05). SSZ and SP decreased basal HMVEC proliferation, while 5-ASA increased proliferation (P<0.05; [n = 5]). SSZ decreased bFGF-induced HMVEC proliferation (P<0.05 [n = 5]). SSZ inhibited phorbol 12-myristate 13-acetate-induced HMVEC tube formation (P<0.05; [minimum n = 5]). Tumor necrosis factor alpha-stimulated HMVEC shedding of sICAM-1 was reduced by incubation with either SSZ (19%) or 5-ASA (23%) (P<0.05; [n = 6]). SP inhibited cytokine-stimulated HMVEC expression of IL-8 and MCP-1 (P<0.05; [n = 4]). Neither SSZ nor its metabolites had any effect on HMVEC production of sE-selectin, GROalpha, or ENA-78. These results demonstrate that SSZ and its metabolite SP may affect the pathogenesis of RA by inhibiting EC chemotaxis, proliferation, tube formation, and expression of sICAM-1, IL-8, and MCP-1.

BackgroundOncostatin M (OSM) is a pleiotropic cytokine, highly expressed in the RA joint that displays both agonistic and antagonistic effects depending on the inflammatory microenvironment. This study examines the effect...

Fibrin and Collagen Differentially Regulate Human Dermal Microvascular Endothelial Cell Integrins: Stabilization of αv/β3 mRNA by Fibrin

To study the effects of an amelogenin mixture on integrin-dependent adhesion, DNA synthesis and apoptosis of cultured human dermal microvascular endothelial cells and angiogenesis in an organotypic assay. Immobilised antibodies against specific integrins (alpha-1, alpha-2, alpha-3, alpha-4, alpha-5, alpha-v, ß1, ß2, ß3, ß4, ß6, alpha-vß3, alpha-vß5 and alpha-5ß1) were used to capture treated human dermal microvascular endothelial cells, which were detected colourimetrically. DNA synthesis of the cells was monitored by 5-bromo-2'- deoxyuridine incorporation and apoptosis by a TdT-mediated dUTP nick-end labelling technique. Tubule formation from aortic arches of 13-d-old chick embryos were followed over 48h. The amelogenin mixture increased microvessel outgrowth by 76% (p < 0.01, n=12) from the aortic explants. Also, amelogenins increased the adhesion (p < 0.01, n = 5) by multiple angiogenesis associated integrin subunits and alpha-vß3, alpha-vß5 and alpha-5ß1 heterodimers on human dermal microvascular endothelial cells at a non-mitogenic concentration (100 µg/ml). Conversely, amelogenins at 1,000 µg/ml decreased microvessel formation possibly due to attenuation of corresponding integrins despite increasing (p < 0.001, n = 8) DNA synthesis. No significant apoptosis was detected in human dermal microvascular endothelial cells cultured on Matrigel with and without amelogenins. Increased surface expression of integrins on endothelial cells may contribute to the proangiogenic property of amelogenins.

miR-199a-5p plays a critical role in controlling cardiomyocyte survival. However, its significance in endothelial cell biology remains ambiguous. Here, we report the first evidence that miR-199a-5p negatively regulates angiogenic responses by directly targeting v-ets erythroblastosis virus E26 oncogene homolog 1 (Ets-1). Induction of miR-199a-5p in human dermal microvascular endothelial cells (HMECs) blocked angiogenic response in Matrigel® culture, whereas miR-199a-5p-deprived cells exhibited enhanced angiogenesis in vitro. Bioinformatics prediction and miR target reporter assay recognized Ets-1 as a novel direct target of miR-199a-5p. Delivery of miR-199a-5p blocked Ets-1 expression in HMECs, whereas knockdown endogenous miR-199a-5p induced Ets-1 expression. Matrix metalloproteinase 1 (MMP-1), one of the Ets-1 downstream mediators, was negatively regulated by miR-199a-5p. Overexpression of Ets-1 not only rescued miR-199a-5p-dependent anti-angiogenic effects but also reversed miR-199a-5p-induced loss of MMP-1 expression. Similarly, Ets-1 knockdown blunted angiogenic response and induction of MMP-1 in miR-199a-5p-deprived HMECs. Examination of cutaneous wound dermal tissue revealed a significant down-regulation of miR-199a-5p expression, which was associated with induction of Ets-1 and MMP-1. Mice carrying homozygous deletions in the Ets-1 gene exhibited blunted wound blood flow and reduced abundance of endothelial cells. Impaired wound angiogenesis was associated with compromised wound closure, insufficient granulation tissue formation, and blunted induction of MMP-1. Thus, down-regulation of miR-199a-5p is involved in the induction of wound angiogenesis through derepressing of the Ets-1-MMP1 pathway.