Endothelial cell calpain as a critical modulator of angiogenesis.

Calpains are a family of calcium-dependent non-lysosomal cysteine proteases. In particular, calpains residing in the endothelial cells play important roles in angiogenesis. It has been shown that calpain activity can be increased in endothelial cells by growth factors, primarily vascular endothelial growth factor (VEGF). VEGF/VEGFR2 induces calpain 2 dependent activation of PI3K/AMPK/Akt/eNOS pathway, and consequent nitric oxide production and physiological angiogenesis. Under pathological conditions such as tumor angiogenesis, endothelial calpains can be activated by hypoxia. This review focuses on the molecular regulatory mechanisms of calpain activation, and the newly identified mechanistic roles and downstream signaling events of calpains in physiological angiogenesis, and in the conditions of pathological tumor angiogenesis and diabetic wound healing, as well as retinopathy and atherosclerosis that are also associated with an increase in calpain activity. Further discussed include the differential strategies of modulating angiogenesis through manipulating calpain expression/activity in different pathological settings. Targeted limitation of angiogenesis in cancer and targeted promotion of angiogenesis in diabetic wound healing via modulations of calpains and calpain-dependent signaling mechanisms are of significant translational potential. Emerging strategies of tissue-specific targeting, environment-dependent targeting, and genome-targeted editing may turn out to be effective regimens for targeted manipulation of angiogenesis through calpain pathways, for differential treatments including both attenuation of tumor angiogenesis and potentiation of diabetic angiogenesis.

Transcription profiles of the ductus arteriosus in Brown-Norway rats with irregular elastic fiber formation.

BACKGROUND: Patent ductus arteriosus (PDA) is one of the most common congenital cardiovascular defects in children. The Brown-Norway (BN) inbred rat presents a higher frequency of PDA. A previous study reported that 2 different quantitative trait loci on chromosomes 8 and 9 were significantly linked to PDA in this strain. Nevertheless, the genetic or molecular mechanisms underlying PDA phenotypes in BN rats have not been fully investigated yet. METHODS AND RESULTS: It was found that the elastic fibers were abundant in the subendothelial area but scarce in the media even in the closed ductus arteriosus (DA) of full-term BN neonates. DNA microarray analysis identified 52 upregulated genes (fold difference >2.5) and 23 downregulated genes (fold difference <0.4) when compared with those of F344 control neonates. Among these genes, 8 (Tbx20, Scn3b, Stac, Sphkap, Trpm8, Rup2, Slc37a2, and RGD1561216) are located in chromosomes 8 and 9. Interestingly, it was also suggested that the significant decrease in the expression levels of the PGE2-specfic receptor, EP4, plays a critical role in elastogenesis in the DA. CONCLUSIONS: BN rats exhibited dysregulation of elastogenesis in the DA. DNA microarray analysis identified the candidate genes including EP4 involved in the DNA phenotype. Further investigation of these newly identified genes will hopefully clarify the molecular mechanisms underlying the irregular formation of elastic fibers in PDA.

Perioperative Changes in Plasma Nitrite and IL-6 Levels Predict Postoperative Atrial Fibrillation (POAF) and Acute Kidney Injury (AKI) after Cardiac Surgery.

Background: Postoperative atrial fibrillation (POAF) and acute kidney injury (AKI) are common yet significant complications after cardiac surgery, with incidences of up to 40% for each. Here, we assessed plasma nitrite and serum interleukin-6 (IL-6) levels before and after cardiac surgery to quantify the extent to which oxidative stress and inflammation contribute to POAF and AKI occurrence. Methods: We prospectively enrolled 206 cardiac surgical patients. Plasma nitrite and serum IL-6 levels were determined preoperatively and at 24 h, 48 h and 72 h postoperatively. The patients had continuous EKG monitoring for occurrence of POAF, while daily serum creatinine was measured for determination of stage 1 + AKI. Results: Postoperatively, 78 (38%) patients experienced AF, and 47 (23%) patients experienced stage 1 + AKI. POAF analysis: Age, ACE-inhibitor use, valve surgery and percent change in baseline plasma nitrite at 24 h postoperatively were associated with POAF in multiple logistic regression analysis. The inclusion of this new biomarker significantly improved the POAF prediction model (AUC 0.77 for clinical risk factors alone, to AUC 0.81). AKI analysis: A history of diabetes mellitus was associated with AKI in multiple logistic regression analysis, and the addition of preoperative IL-6 levels improved the prediction model for AKI occurrence (AUC 0.69 to AUC 0.74). Conclusions: We previously observed selective upregulation of NADPH oxidase isoform 4 (NOX4) in patients with AF, a critical causal role of NOX4 for AF in zebrafish and a robust inhibitory effect of nitric oxide (NO) on NOX4. Our data innovatively demonstrate that a reduction in circulating nitrite levels, likely implicative of elevated NOX4-mediated oxidative stress, independently associates with POAF and improves POAF prediction, whereas the inclusion of circulating IL-6 levels improves the prediction model for AKI. Therefore, therapeutic strategies to mitigate these pathophysiological sequalae of surgical stress may reduce the incidence of severe postoperative complications of POAF and AKI.

Targeting MicroRNA-192-5p, a Downstream Effector of NOXs (NADPH Oxidases), Reverses Endothelial DHFR (Dihydrofolate Reductase) Deficiency to Attenuate Abdominal Aortic Aneurysm Formation.

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Attenuation of neointimal formation with netrin-1 and netrin-1 preconditioned endothelial progenitor cells.

Restenosis after angioplasty is a serious clinical problem that can result in re-occlusion of the coronary artery. Although current drug-eluting stents have proved to be more effective in reducing restenosis, they have drawbacks of inhibiting reendothelialization to promote thrombosis. New treatment options are in urgent need. We have shown that netrin-1, an axon-guiding protein, promotes angiogenesis and cardioprotection via production of nitric oxide (NO). The present study examined whether and how netrin-1 attenuates neointimal formation in a femoral wire injury model. Infusion of netrin-1 into C57BL/6 mice markedly attenuated neointimal formation following wire injury of femoral arteries, measured by intimal to media ratio (from 1.94 ± 0.55 to 0.45 ± 0.86 at 4 weeks). Proliferation of VSMC in situ was largely reduced. This protective effect was absent in DCC+/- animals. NO production was increased by netrin-1 in both intact and injured femoral arteries, indicating netrin-1 stimulation of endogenous NO production from intact endothelium and remaining endothelial cells post-injury. VSMC migration was abrogated by netrin-1 via a NO/cGMP/p38 MAPK pathway, while timely EPC homing was induced. Injection of netrin-1 preconditioned wild-type EPCs, but not EPCs of DCC+/- animals, substantially attenuated neointimal formation. EPC proliferation, NO production, and resistance to oxidative stress induced apoptosis were augmented by netrin-1 treatment. In conclusion, our data for the first time demonstrate that netrin-1 is highly effective in reducing neointimal formation following vascular endothelial injury, which is dependent on DCC, and attributed to inhibition of VSMC proliferation and migration, as well as improved EPC function. These data may support usage of netrin-1 and netrin-1 preconditioned EPCs as novel therapies for post angioplasty restenosis. KEY MESSAGE: Netrin-1 attenuates neointimal formation following post endothelial injury via DCC and NO. Netrin-1 inhibits VSMC proliferation in situ following endothelial injury. Netrin-1 inhibits VSMC migration via a NO/cGMP/p38 MAPK pathway. Netrin-1 augments proliferation of endothelial progenitor cells (EPCs) and EPC eNOS/NO activation. Netrin-1 enhances resistance of EPCs to oxidative stress, improving re-endothelialization following injury.

Thromboxane A(2) receptor stimulation promotes closure of the rat ductus arteriosus through enhancing neointima formation.

Ductus arteriosus (DA) closure follows constriction and remodeling of the entire vessel wall. Patent ductus arteriosus occurs when the DA does not close after birth, and this condition is currently treated using cyclooxygenase inhibitors. However, the efficacy of cyclooxygenase inhibitors is often limited. Our previous study demonstrated that low-dose thromboxane A2 receptor (TP) stimulation constricted the DA with minimal adverse effects in rat neonates. However, its effect on DA remodeling remains unknown. In this study, we focused on the impact of the exogenous TP stimulation on the DA remodeling, especially intimal thickening. Using DA explants from rat fetuses at embryonic day 19 as a ex vivo model and primary cultured rat DA smooth muscle cells from embryonic day 21 as a in vitro model, we evaluated the effect of TP stimulation on the DA remodeling. The selective TP agonists U46619 and I-BOP promoted neointima formation in the ex vivo DA explants, and TP stimulation increased DA SMC migration in a dose-dependent manner. Both effects were inhibited by the selective TP antagonist SQ29548 or the siRNA against TP. TP stimulation also increased DA SMC proliferation in the presence of 10% fetal bovine serum. LC/MS/MS analysis revealed that TP stimulation increased secretion of several extracellular matrix proteins that may contribute to an increase in neointima formation. In conclusion, we uncovered that exogenous administration of TP agonist promotes neointima formation through the induction of migration and proliferation of DA SMC, which could contribute to DA closure and also to its vasoconstrictive action.

Transcription profiles of endothelial cells in the rat ductus arteriosus during a perinatal period.

Endothelial cells (ECs) lining the blood vessels serve a variety of functions and play a central role in the homeostasis of the circulatory system. Since the ductus arteriosus (DA) has different arterial characteristics from its connecting vessels, we hypothesized that ECs of the DA exhibited a unique gene profile involved in the regulation of DA-specific morphology and function. Using a fluorescence-activated cell sorter, we isolated ECs from pooled tissues from the DA or the descending aorta of Wistar rat fetuses at full-term of gestation (F group) or neonates 30 minutes after birth (N group). Using anti-CD31 and anti-CD45 antibodies as cell surface markers for ECs and hematopoietic derived cells, respectively, cDNAs from the CD31-positive and CD45-negative cells were hybridized to the Affymetrix GeneChip® Rat Gene 1.0 ST Array. Among 26,469 gene-level probe sets, 82 genes in the F group and 81 genes in the N group were expressed at higher levels in DA ECs than in aortic ECs (p<0.05, fold change>2.0). In addition to well-known endothelium-enriched genes such as Tgfb2 and Vegfa, novel DA endothelium-dominant genes including Slc38a1, Capn6, and Lrat were discovered. Enrichment analysis using GeneGo MetaCore software showed that DA endothelium-related biological processes were involved in morphogenesis and development. We identified many overlapping genes in each process including neural crest-related genes (Hoxa1, Hoxa4, and Hand2, etc) and the second heart field-related genes (Tbx1, Isl1, and Fgf10, etc). Moreover, we found that regulation of epithelial-to-mesenchymal transition, cell adhesion, and retinol metabolism are the active pathways involved in the network via potential interactions with many of the identified genes to form DA-specific endothelia. In conclusion, the present study uncovered several significant differences of the transcriptional profile between the DA and aortic ECs. Newly identified DA endothelium-dominant genes may play an important role in DA-specific functional and morphologic characteristics.