MicroRNA-216a is essential for cardiac angiogenesis.

While it is experimentally supported that impaired myocardial vascularization contributes to a mismatch between myocardial oxygen demand and supply, a mechanistic basis for disruption of coordinated tissue growth and angiogenesis in heart failure remains poorly understood. Silencing strategies that impair microRNA biogenesis have firmly implicated microRNAs in the regulation of angiogenesis, and individual microRNAs prove to be crucial in developmental or tumor angiogenesis. A high-throughput functional screening for the analysis of a whole-genome microRNA silencing library with regard to their phenotypic effect on endothelial cell proliferation as a key parameter, revealed several anti- and pro-proliferative microRNAs. Among those was miR-216a, a pro-angiogenic microRNA which is enriched in cardiac microvascular endothelial cells and reduced in expression under cardiac stress conditions. miR-216a null mice display dramatic cardiac phenotypes related to impaired myocardial vascularization and unbalanced autophagy and inflammation, supporting a model where microRNA regulation of microvascularization impacts the cardiac response to stress.

Therapeutic Delivery of miR-148a Suppresses Ventricular Dilation in Heart Failure.

Heart failure is preceded by ventricular remodeling, changes in left ventricular mass, and myocardial volume after alterations in loading conditions. Concentric hypertrophy arises after pressure overload, involves wall thickening, and forms a substrate for diastolic dysfunction. Eccentric hypertrophy develops in volume overload conditions and leads wall thinning, chamber dilation, and reduced ejection fraction. The molecular events underlying these distinct forms of cardiac remodeling are poorly understood. Here, we demonstrate that miR-148a expression changes dynamically in distinct subtypes of heart failure: while it is elevated in concentric hypertrophy, it decreased in dilated cardiomyopathy. In line, antagomir-mediated silencing of miR-148a caused wall thinning, chamber dilation, increased left ventricle volume, and reduced ejection fraction. Additionally, adeno-associated viral delivery of miR-148a protected the mouse heart from pressure-overload-induced systolic dysfunction by preventing the transition of concentric hypertrophic remodeling toward dilation. Mechanistically, miR-148a targets the cytokine co-receptor glycoprotein 130 (gp130) and connects cardiomyocyte responsiveness to extracellular cytokines by modulating the Stat3 signaling. These findings show the ability of miR-148a to prevent the transition of pressure-overload induced concentric hypertrophic remodeling toward eccentric hypertrophy and dilated cardiomyopathy and provide evidence for the existence of separate molecular programs inducing distinct forms of myocardial remodeling.

Human cytomegalovirus infection impairs endothelial cell chemotaxis by disturbing VEGF signalling and actin polymerization.

AIMS: Human cytomegalovirus (HCMV) infection has been linked to the pathogenesis of vasculopathies; however, its pathogenic relevance remains to be established. A prerequisite for vascular repair is endothelial cell migration. We evaluated the influence of HCMV on chemokinesis and chemotactic response of human coronary artery endothelial cells (HCAEC) towards vascular endothelial growth factor (VEGF). METHODS AND RESULTS: A virus dose-dependent reduction in chemokinesis and VEGF-dependent chemotaxis was observed (P < 0.05). UV-inactivated virus did not inhibit chemotaxis or chemokinesis, indicating that viral gene expression is mandatory. We identified two HCMV-induced mechanisms explaining the reduction of chemotaxis: first, a non-ambiguous reduction of VEGFR-2 protein was observed, due to decreased transcription. This protein down-modulation could not be inhibited by Ganciclovir. The remaining VEGFR-2 expressed on infected HCAEC was able to stimulate cell activation. Second, HCMV infection influences actin polymerization in HCAEC as shown by FACS analysis: actin polymerization was significantly reduced to 53 and 51% (P < 0.05) compared with non-infected HCAEC at 24 and 72 h p.i., respectively. Genetically and pharmacologically eliminated VEGFR-2 function resulted in a significant (P < 0.05) reduction of VEGF-induced activation of actin polymerization. CONCLUSION: We demonstrated a significant reduction of the chemotactic mobility of HCMV-infected HCAEC mediated by down-modulation of the VEGFR-2 and by inhibition of actin polymerization. This VEGF resistance of HCMV-infected endothelial cells is likely to promote atherogenesis.

TGF-ß1/ALK5-induced monocyte migration involves PI3K and p38 pathways and is not negatively affected by diabetes mellitus.

AIMS: Monocytes contribute to arteriogenesis by infiltration to sites of collateral growth and subsequent production and release of growth factors. Transforming growth factor ß1 (TGF-ß1) mediates monocyte motility and stimulates arteriogenesis. TGF-ß1 signalling mechanisms mediating monocyte motility are unknown so far. Moreover, the influence of cardiovascular risk factor diabetes on TGF-ß1-induced monocyte migration remains to be elucidated. METHODS AND RESULTS: Stimulation of primary human monocytes with TGF-ß1 endorsed phosphorylation of v-Akt murine thymoma viral oncogene analogues protein (AKT), p38, and extracellular signal-related kinase 1/2 (ERK1/2), besides the activation of the SMA/MAD homologues protein (SMAD) pathway. Inhibition of the TGF-ßtype 1 receptor, alias activin receptor-like kinase 5 (ALK5), hindered monocyte chemotaxis towards TGF-ß1 and TGF-ß1-activated downstream signalling cascades. Individual genetic knock-downs for receptor-regulated SMAD2 and SMAD3 did not affect monocyte migration to TGF-ß1. Inhibition of phosphoinositide 3 kinase (PI3K) activity, but not AKT, diminished both basal and TGF-ß1-mediated monocyte motility. TGF-ß1-induced monocyte chemotaxis did not rely on ERK1/2, but rather on p38. Remarkably, TGF-ß1 was able to stimulate chemotaxis of diabetic monocytes. CONCLUSION: The current study provides novel insights into the molecular mechanisms of TGF-ß1-induced monocyte migration, requiring ALK5 kinase activity and signalling via PI3K and p38. TGF-ß1-driven monocyte motogenicity is fully functional in diabetic conditions, which is in sharp contrast to the impaired chemotactic responses to certain other arteriogenic cytokines. Therefore, TGF-ß1 may be a promising candidate for endogenously and exogenously stimulating collateral growth in diabetic patients.

VEGF-A-induced chemotaxis of CD16+ monocytes is decreased secondary to lower VEGFR-1 expression.

BACKGROUND: Monocyte recruitment into the vessel wall is a crucial initial step in vascular repair, arteriogenesis and atherogenesis. Two distinct human monocyte subpopulations can be classified according to their CD14/16 surface expression, namely CD14++CD16-monocytes (CD16-mo) and CD14+CD16+ monocytes (CD16+mo). We investigated different functional properties of the two monocyte subsets. METHODS: CD16-/CD16+mo were isolated from human blood by an immunological selection. We assessed monocyte chemokinesis, chemotaxis, adhesion and Vascular-Endothelial Growth Factor (VEGF) receptor expression. Furthermore, generation of reactive oxygen species (ROS) as well as expression of antioxidant enzymes was investigated. RESULTS: Chemokinesis of CD16+mo was decreased compared to CD16-mo (p<0.01). Likewise, adhesion capacity of CD16+mo was weaker (p<0.05). CD16+mo chemotaxis towards the angiogenic ligands vascular endothelial growth factor-A (VEGF-A) and placenta growth factor-1 (PlGF-1) was reduced compared to CD16-mo. VEGFR-1 is the receptor for VEGF-A and PlGF-1 on monocytes. VEGFR-1 protein expression was lower in CD16+mo than in CD16-mo (p<0.05). The impaired VEGF-A- and PlGF-1-induced CD16+mo chemotaxis might therefore be attributed to the reduced VEGFR-1 expression. CD16+mo exhibited less spontaneous ROS production than CD16-mo. Additionally, the antioxidant enzyme manganese superoxide dismutase was expressed at higher levels in CD16+mo (p<0.05); this might partly explain the higher oxidative resistance of CD16+mo. CONCLUSION: These novel functional differences between CD16-mo and CD16+mo may predict different functional roles of both monocyte subsets in vascular repair, arteriogenesis and atherogenesis.

Strenuous physical exercise adversely affects monocyte chemotaxis.

Physical exercise is important for proper cardiovascular function and disease prevention, but it may influence the immune system. We evaluated the effect of strenuous exercise on monocyte chemotaxis. Monocytes were isolated from blood of 13 young, healthy, sedentary individuals participating in a three-week training program which consisted of repeated exercise bouts. Monocyte chemotaxis and serological biomarkers were investigated at baseline, after three weeks training and after four weeks recovery. Chemotaxis towards vascular endothelial growth factor-A (VEGF-A) and transforming growth factor-ß1 (TGF-ß1) was completely inhibited immediately after training (p<0.01), and remained so after four weeks recovery. Likewise, monocyte chemoattractant protein-1 (MCP-1)-induced migration declined after training (p<0.01) and improved only partially during the recovery period. MCP-1 serum levels were significantly reduced after four weeks recovery compared to baseline (p<0.01). Total blood antioxidant capacity was enhanced at this time point (p<0.01). Monocyte chemokinesis, TGF-ß1 and nitric oxide serum levels remained unchanged during the study. Strenuous three-week training consisting of repeated exercise bouts in healthy, sedentary individuals reduces monocyte chemotaxis. It remains to be established, whether this is a sound adaptation to increased stimuli or an untoward reaction to overtraining. Nevertheless, the effect remains for several weeks with no exercise.

Diabetes mellitus activates signal transduction pathways resulting in vascular endothelial growth factor resistance of human monocytes.

BACKGROUND: Monocytes are cellular components of wound repair, arteriogenesis, and atherogenesis. Vascular endothelial growth factor (VEGF)-A and placental growth factor recruit monocytes to sites of arteriogenesis via stimulation of VEGF receptor-1 (VEGFR-1). The chemotactic response of monocytes to VEGF-A is attenuated in individuals with diabetes mellitus (DM). This VEGF resistance correlates with impaired collateral growth. The aim of this study is to elucidate the molecular basis of VEGF resistance and impaired monocyte response in DM. METHODS AND RESULTS: Phosphorylation of Akt, p38, and extracellular signal-regulated kinase 1/2 (ERK1/2) could be stimulated with either placental growth factor-1 or VEGF-A in monocytes from non-DM but not DM individuals. In contrast, formyl-methionyl-leucyl-phenylalanine caused a comparable activation of these molecules in both DM and non-DM monocytes. Baseline phosphorylation of Akt, p38, and ERK1/2 was significantly elevated in monocytes from DM compared with non-DM subjects. Of note, H(2)O(2) activated Akt, p38, and ERK1/2 in non-DM monocytes ex vivo. Protein tyrosine phosphatases had stronger oxidative modifications in monocytes from DM than from non-DM individuals, which reflects functional protein tyrosine phosphatase inhibition, similar to that seen after H(2)O(2) challenge. Overall, protein tyrosine phosphatase and protein tyrosine phosphatase-1B activity were reduced in DM monocytes. DM monocytes revealed higher expression of the receptor for advanced glycation end products. Stimulation with advanced glycation end products ligands resulted in activation of non-DM monocytes and inhibition of VEGFR-1-mediated chemotaxis. The elevated baseline phosphorylation/activation of Akt, p38, and ERK1/2 in DM monocytes likely causes the resistance to further stimulation with specific stimuli such as VEGF-A, revealing a molecular explanation of the DM-related signal transduction defect. CONCLUSIONS: We propose that elevated advanced glycation end products expression and increased oxidative stress in diabetic monocytes lead to activation of VEGFR-1-related signaling pathways and to desensitization of VEGFR-1 responses. These data establish VEGF resistance as a novel molecular concept for DM-related cellular dysfunction.