Amplification of the gene encoding the ErbB2 (Her2/neu) receptor tyrosine kinase is critical for the progression of several forms of breast cancer. In a large-scale clinical trial, treatment with Herceptin (trastuzumab), a humanized blocking antibody against ErbB2, led to marked improvement in survival. However, cardiomyopathy was uncovered as a mitigating side effect, thereby suggesting an important role for ErbB2 signaling as a modifier of human heart failure. To investigate the physiological role of ErbB2 signaling in the adult heart, we generated mice with a ventricular-restricted deletion of Erbb2. These ErbB2-deficient conditional mutant mice were viable and displayed no overt phenotype. However, physiological analysis revealed the onset of multiple independent parameters of dilated cardiomyopathy, including chamber dilation, wall thinning and decreased contractility. Additionally, cardiomyocytes isolated from these conditional mutants were more susceptible to anthracycline toxicity. ErbB2 signaling in cardiomyocytes is therefore essential for the prevention of dilated cardiomyopathy.

Caveolins are important components of caveolae, which have been implicated in vesicular trafficking and signal transduction. To investigate the in vivo significance of Caveolins in mammals, we generated mice deficient in the caveolin-1 (cav-1) gene and have shown that, in the absence of Cav-1, no caveolae structures were observed in several nonmuscle cell types. Although cav-1 ؊/؊ mice are viable, histological examination and echocardiography identified a spectrum of characteristics of dilated cardiomyopathy in the left ventricular chamber of the cav-1-deficient hearts, including an enlarged ventricular chamber diameter, thin posterior wall, and decreased contractility. These animals also have marked right ventricular hypertrophy, suggesting a chronic increase in pulmonary artery pressure. Direct measurement of pulmonary artery pressure and histological analysis revealed that the cav-1 ؊/؊ mice exhibit pulmonary hypertension, which may contribute to the right ventricle hypertrophy. In addition, the loss of Cav-1 leads to a dramatic increase in systemic NO levels. Our studies provided in vivo evidence that cav-1 is essential for the control of systemic NO levels and normal cardiopulmonary function.

Pulmonary hypertension (PH) is an unremitting disease defined by a progressive increase in pulmonary vascular resistance leading to right-sided heart failure. Using mice with genetic deletions of caveolin 1 (Cav1) and eNOS (Nos3), we demonstrate here that chronic eNOS activation secondary to loss of caveolin-1 can lead to PH. Consistent with a role for eNOS in the pathogenesis of PH, the pulmonary vascular remodeling and PH phenotype of Cav1 -/-mice were absent in Cav1 -/-Nos3 -/-mice. Further, treatment of Cav1 -/-mice with either MnTMPyP (a superoxide scavenger) or l-NAME (a NOS inhibitor) reversed their pulmonary vascular pathology and PH phenotype. Activation of eNOS in Cav1 -/-lungs led to the impairment of PKG activity through tyrosine nitration. Moreover, the PH phenotype in Cav1 -/-lungs could be rescued by overexpression of PKG-1. The clinical relevance of the data was indicated by the observation that lung tissue from patients with idiopathic pulmonary arterial hypertension demonstrated increased eNOS activation and PKG nitration and reduced caveolin-1 expression. Together, these data show that loss of caveolin-1 leads to hyperactive eNOS and subsequent tyrosine nitration-dependent impairment of PKG activity, which results in PH. Thus, targeting of PKG nitration represents a potential novel therapeutic strategy for the treatment of PH.

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Background Vascular occlusion and complex plexiform lesions are hallmarks of the pathology of severe pulmonary arterial hypertension (PAH) in patients. However, mechanisms of obliterative vascular remodeling remain elusive and hence current therapies have not targeted the fundamental disease modifying mechanisms and result in only modest improvement in morbidity and mortality. Methods and Results Mice with Tie2Cre-mediated disruption of Egln1 (encoding prolyl-4 hydroxylase 2, PHD2) (Egln1Tie2) in endothelial cells (ECs) and hematopoietic cells exhibited spontaneous severe PAH with extensive pulmonary vascular remodeling including vascular occlusion and plexiform-like lesions resembling the hallmarks of the pathology of clinical PAH. As seen in idiopathic PAH patients, Egln1Tie2 mice exhibited unprecedented right ventricular hypertrophy and failure and progressive mortality. Consistently, PHD2 expression was diminished in lung ECs of obliterated pulmonary vessels in idiopathic PAH patients. Genetic deletions of both Egln1 and Hif1a or Egln1 and Hif2a identified hypoxia-inducible factor-2α (HIF-2α) as the critical mediator of severe PAH seen in Egln1Tie2 mice. We also observed altered expression of many PH-causing genes in Egln1Tie2 lungs which was also normalized in Egln1Tie2/Hif2aTie2 lungs. PHD2-deficient ECs promoted smooth muscle cell proliferation in part through HIF-2α-activated CXCL12 expression. Genetic deletion of Cxcl12 attenuated PAH in Egln1Tie2 mice. Conclusions These studies defined an unexpected role of PHD2 deficiency in the mechanisms of severe PAH and identified the first genetically modified mouse model with obliterative vascular remodeling and pathophysiology recapitulating clinical PAH. Thus, targeting PHD2/HIF-2α signaling is a promising strategy to reverse vascular remodeling for treatment of severe PAH.

Caveolin‐1 (Cav‐1), the principal structural protein of caveolae, is implicated in normal endothelial barrier function, buts its role in lung inflammation is not well understood. Using caveolin‐1 knockout (Cav‐1 −/−) mice, we addressed the role of Cav‐1 in sepsis‐induced lung injury. We assessed lung inflammation in Cav‐1 −/− mice following i.p. injection of LPS. Neutrophil (PMN) binding was measured by an adhesion assay performed on primary cultured mouse endothelial cells. PMN sequestration was assessed by myeloperoxidase activity in the whole lung. Lung microvascular permeability was determined using iodine‐125 radio‐labeled albumin. When compared to wild type (WT) mice, Cav‐1 −/− mice exhibited significantly impaired PMN binding and sequestration after LPS challenge. LPS‐induced increases in lung microvascular permeability and edema formation were also markedly reduced in Cav‐1 −/− mice relative to WT. To address the basis of the reduced lung inflammation, we examined the specific role of nitric oxide (NO). As Cav‐1 is known to sequester eNOS rendering it inactive, we postulated that in Cav‐1 −/− mice there should be increased eNOS activity and eNOS‐derived NO levels in response to LPS challenge. We observed a marked increase in eNOS activity and NO production in Cav‐1 −/− lung relative to WT. Corresponding to the known role of NO in modulating NF‐κB activity, we also noted a time‐dependent suppression of NF‐κB activity in Cav‐1 −/− mice. Thus, Cav‐1 expression and its ability to regulate eNOS‐derived NO production is a crucial determinant of the lung inflammatory response to sepsis. This abstract is funded by: NIH(T32HL007829, HL60678, HL77806)

Neuregulins (i.e. neuregulin-1 (NRG1), also called neu differentiation factor, heregulin, glial growth factor, and acetylcholine receptor-inducing activity) are known to induce growth and differentiation of epithelial, glial, neuronal, and skeletal muscle cells. Unexpectedly, mice with loss of function mutations of NRG1 or of either of two of their cognate receptors, ErbB2 and ErbB4, die during midembryogenesis due to the aborted development of myocardial trabeculae in ventricular muscle. To examine the role of NRG and their receptors in developing and postnatal myocardium, we studied the ability of a soluble NRG1 (recombinant human glial growth factor 2) to promote proliferation, survival, and growth of isolated neonatal and adult rat cardiac myocytes. Both ErbB2 and ErbB4 receptors were found to be expressed by neonatal and adult ventricular myocytes and activated by rhGGF2. rhGGF2 (30 ng/ml) provoked an approximate 2-fold increase in embryonic cardiac myocyte proliferation. rhGGF2 also promoted survival and inhibited apoptosis of subconfluent, serum-deprived myocyte primary cultures and also induced hypertrophic growth in both neonatal and adult ventricular myocytes, which was accompanied by enhanced expression of prepro-atrial natriuretic factor and skeletal ␣-actin. Moreover, NRG1 mRNA could be detected in coronary microvascular endothelial cell primary cultures prepared from adult rat ventricular muscle. NRG1 expression in these cells was increased by endothelin-1, another locally acting cardiotropic peptide within the heart. The persistent expression of both a neuregulin and its cognate receptors in the postnatal and adult heart suggests a continuing role for neuregulins in the myocardial adaption to physiologic stress or injury.

Background The blood-brain barrier (BBB) formed by brain endothelial cells (ECs) interconnected by tight junctions (TJs) is essential for the homeostasis of the central nervous system (CNS). Although studies have shown the importance of various signaling molecules in BBB formation during development, little is known about the molecular basis regulating the integrity of the adult BBB. Methods and Results Using a mouse model with tamoxifen-inducible EC-restricted disruption of ctnnb1 (iCKO), here we show that endothelial β-catenin signaling is essential for maintaining BBB integrity and CNS homeostasis in adult. The iCKO mice developed severe seizures accompanied by neuronal injury, multiple brain petechial hemorrhages, and CNS inflammation, and all died postictal. Disruption of endothelial β-catenin induced BBB breakdown and downregulation of specific TJ proteins Claudin-1 and -3 in adult brain ECs. The clinical relevance of the data is indicated by the observation of decreased expression of Claudin-1 and nuclear β-catenin in brain ECs of hemorrhagic lesions of hemorrhagic stroke patients. Conclusion These results demonstrate the prerequisite role of endothelial β-catenin in maintaining the integrity of adult BBB. The results suggest that BBB dysfunction secondary to defective β-catenin transcription activity is a key pathogenic factor in hemorrhagic stroke, seizure activity and CNS inflammation.