CaMK4 Gene Deletion Induces Hypertension.

BACKGROUND: The expression of calcium/calmodulin-dependent kinase IV (CaMKIV) was hitherto thought to be confined to the nervous system. However, a recent genome-wide analysis indicated an association between hypertension and a single-nucleotide polymorphism (rs10491334) of the human CaMKIV gene (CaMK4), which suggests a role for this kinase in the regulation of vascular tone. METHODS AND RESULTS: To directly assess the role of CaMKIV in hypertension, we characterized the cardiovascular phenotype of CaMK4(-/-) mice. They displayed a typical hypertensive phenotype, including high blood pressure levels, cardiac hypertrophy, vascular and kidney damage, and reduced tolerance to chronic ischemia and myocardial infarction compared with wild-type littermates. Interestingly, in vitro experiments showed the ability of this kinase to activate endothelial nitric oxide synthase. Eventually, in a population study, we found that the rs10491334 variant associates with a reduction in the expression levels of CaMKIV in lymphocytes from hypertensive patients. CONCLUSIONS: Taken together, our results provide evidence that CaMKIV plays a pivotal role in blood pressure regulation through the control of endothelial nitric oxide synthase activity. (J Am Heart Assoc. 2012;1:e001081 doi: 10.1161/JAHA.112.001081.).

Endothelial cells are able to synthesize and release catecholamines both in vitro and in vivo.

Recently it has been demonstrated that catecholamines are produced and used by macrophages and mediate immune response. The aim of this study is to verify whether endothelial cells (ECs), which are of myeloid origin, can produce catecholamines. We demonstrated that genes coding for tyrosine hydroxylase, Dopa decarboxylase, dopamine ß hydroxylase (DßH), and phenylethanolamine-N-methyl transferase, enzymes involved in the synthesis of catecholamines, are all expressed in basal conditions in bovine aorta ECs, and their expression is enhanced in response to hypoxia. Moreover, hypoxia enhances catecholamine release. To evaluate the signal transduction pathway that regulates catecholamine synthesis in ECs, we overexpressed in bovine aorta ECs either protein kinase A (PKA) or the transcription factor cAMP response element binding, because PKA/cAMP response element binding activation induces tyrosine hydroxylase transcription and activity in response to stress. Both cAMP response element binding and PKA overexpression enhance DßH and phenylethanolamine-N-methyl transferase gene expression and catecholamine release, whereas H89, inhibitor of PKA, exerts the opposite effect, evidencing the role of PKA/cAMP response element binding transduction pathway in the regulation of catecholamine release in bovine aorta ECs. We then evaluated by immunohistochemistry the expression of tyrosine hydroxylase, Dopa decarboxylase, DßH, and phenylethanolamine-N-methyl transferase in femoral arteries from hindlimbs of C57Bl/6 mice 3 days after removal of the common femoral artery to induce chronic ischemia. Ischemia evokes tyrosine hydroxylase, Dopa decarboxylase, DßH, and phenylethanolamine-N-methyl transferase expression in the endothelium. Finally, the pharmacological inhibition of catecholamine release by fusaric acid, an inhibitor of DßH, reduces the ability of ECs to form network-like structures on Matrigel matrix. In conclusion, our study demonstrates for the first time that ECs are able to synthesize and release catecholamines in response to ischemia.

Age-related impairment in insulin release: the essential role of ß(2)-adrenergic receptor.

In this study, we investigated the significance of ß(2)-adrenergic receptor (ß(2)AR) in age-related impaired insulin secretion and glucose homeostasis. We characterized the metabolic phenotype of ß(2)AR-null C57Bl/6N mice (ß(2)AR(-/-)) by performing in vivo and ex vivo experiments. In vitro assays in cultured INS-1E ß-cells were carried out in order to clarify the mechanism by which ß(2)AR deficiency affects glucose metabolism. Adult ß(2)AR(-/-) mice featured glucose intolerance, and pancreatic islets isolated from these animals displayed impaired glucose-induced insulin release, accompanied by reduced expression of peroxisome proliferator-activated receptor (PPAR)γ, pancreatic duodenal homeobox-1 (PDX-1), and GLUT2. Adenovirus-mediated gene transfer of human ß(2)AR rescued these defects. Consistent effects were evoked in vitro both upon ß(2)AR knockdown and pharmacologic treatment. Interestingly, with aging, wild-type (ß(2)AR(+/+)) littermates developed impaired insulin secretion and glucose tolerance. Moreover, islets from 20-month-old ß(2)AR(+/+) mice exhibited reduced density of ß(2)AR compared with those from younger animals, paralleled by decreased levels of PPARγ, PDX-1, and GLUT2. Overexpression of ß(2)AR in aged mice rescued glucose intolerance and insulin release both in vivo and ex vivo, restoring PPARγ/PDX-1/GLUT2 levels. Our data indicate that reduced ß(2)AR expression contributes to the age-related decline of glucose tolerance in mice.

GRK2 levels in umbilical arteries of pregnancies complicated by gestational hypertension and preeclampsia.

BACKGROUND: G-Protein coupled receptor kinase 2 (GRK2) represents a regulator of cell function in different cardiovascular conditions, including high blood pressure. The relationship between elevated GRK2 levels and impaired vasorelaxant responses is causative of hypertension through the increase in vascular resistances. The aim of this study is to ascertain if this feature is present in the fetal placental vasculature of pregnancies complicated by hypertensive disorders. METHODS: We have assessed GRK2 levels in the umbilical arteries (UA) of 21 preeclamptic or gestational hypertensive and 23 normotensive women at time of delivery. RESULTS: GRK2 levels were increased in the hypertensive group (0.83 ± 0.14 vs. 0.48 ± 0.06 densitometry units; P < 0.05). GRK2 levels were in correlation and in linear regression with systolic, diastolic, and mean arterial pressure (P < 0.05, r(2) = 0.12, r(2) = 0.11, r(2) = 0.12). Correlations did not reach a significant value for other clinical parameters such as gestational age at birth, umbilical artery pulsatility index, maternal proteinuria, and neonatal birth weight. Out of the 21 hypertensive women, 7 who developed a preeclampsia associated with early preterm delivery (before 34 weeks) had a significantly lower GRK2 levels compared to the remaining 14 (0.51 ± 0.12 vs. 1.08 ± 0.20 densitometry units, P < 0.05). CONCLUSIONS: We conclude that elevated GRK2 levels in the umbilical vasculature is correlated to elevated blood pressure levels, with a likely compensatory rather than causative role since the lack of protective effect of elevated GRK2 levels may negatively affect the outcome of the hypertensive state.

Evaluation of the anti-angiogenic properties of the new selective αVß3 integrin antagonist RGDechiHCit.

BACKGROUND: Integrins are heterodimeric receptors that play a critical role in cell-cell and cell-matrix adhesion processes. Among them, αVß3 integrin, that recognizes the aminoacidic RGD triad, is reported to be involved in angiogenesis, tissue repair and tumor growth. We have recently synthesized a new and selective ligand of αVß3 receptor, referred to as RGDechiHCit, that contains a cyclic RGD motif and two echistatin moieties. METHODS: The aim of this study is to evaluate in vitro and in vivo the effects of RGDechiHCit. Therefore, we assessed its properties in cellular (endothelial cells [EC], and vascular smooth muscle cells [VSMC]) and animal models (Wistar Kyoto rats and c57Bl/6 mice) of angiogenesis. RESULTS: In EC, but not VSMC, RGDechiHCit inhibits intracellular mitogenic signaling and cell proliferation. Furthermore, RGDechiHCit blocks the ability of EC to form tubes on Matrigel. In vivo, wound healing is delayed in presence of RGDechiHCit. Similarly, Matrigel plugs demonstrate an antiangiogenic effect of RGDechiHCit. CONCLUSIONS: Our data indicate the importance of RGDechiHCit in the selective inhibition of endothelial αVß3 integrin in vitro and in vivo. Such inhibition opens new fields of investigation on the mechanisms of angiogenesis, offering clinical implications for treatment of pathophysiological conditions such as cancer, proliferative retinopathy and inflammatory disease.

Intracardiac injection of AdGRK5-NT reduces left ventricular hypertrophy by inhibiting NF-kappaB-dependent hypertrophic gene expression.

Several studies underline the role of the transcription factor NF-κB in the development of left cardiac hypertrophy (LVH). We have demonstrated recently that the RGS homology domain within the amino terminus of GRK5 (GRK5-NT) is able to inhibit NF-κB transcription activity and its associated phenotypes. The aim of this study was to evaluate the ability of GRK5-NT to regulate LVH through the inhibition of NF-κB both in vitro and in vivo. In cardiomyoblasts, GRK5-NT inhibits phenylephrine-induced transcription of both NF-κB and atrial natriuretic factor promoters, assessed by luciferase assay, thus confirming a role for this protein in the regulation of cardiomyocyte hypertrophy. In vivo, we explored 2 rat models of LVH, the spontaneously hypertensive rat and the normotensive Wistar Kyoto rat exposed to chronic administration of phenylephrine. Intracardiac injection of an adenovirus encoding for GRK5-NT reduces cardiac mass in spontaneously hypertensive rats and prevents the development of phenylephrine-induced LVH in Wistar Kyoto rats. This associates with inhibition of NF-κB signaling (assessed by NF-κB levels), transcriptional activity and phenotypes (fibrosis and apoptosis). Such phenomenon is independent from hemodynamic changes, because adenovirus encoding for GRK5-NT did not reduce blood pressure levels in spontaneously hypertensive rats or in Wistar Kyoto rats. In conclusion, our study supports the regulation of LVH based on the GRK5-NT inhibition of the NF-κB transduction signaling.