Objective-Rho-Kinase activity is increased in cardiovascular diseases and in patients with cardiovascular risk factors.However, it is not known whether inhibition of Rho-kinase could lead to cardiovascular protection and, if so, by what mechanism. Methods and Results-In human endothelial cells, the Rho-kinase inhibitor, hydroxyfasudil (HF) (1 to 100 mol/L), increased Akt serine-473 phosphorylation within 15 minutes, leading to a 2.2-fold and 4.0-fold increase in Akt kinase activity and nitric oxide (NO) release, respectively. Activation of Akt and eNOS by HF was completely blocked by the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, LY294002 (10 mol/L). To determine the physiological relevance of this pathway, we used 2 models of ischemia-reperfusion (I/R) injury. Acute administration of fasudil (10 mg/kg, intraperitoneal, 1 hour before ischemia) decreased leukocyte recruitment and adhesion to the mesenteric endothelium after I/R injury in wild-type but not eNOS Ϫ/Ϫ mice. Similarly, treatment with fasudil decreased myocardial infarct size by 38% in rats subjected to transient coronary artery occlusion. Cotreatment with 2 PI3-kinase inhibitors, wortmannin and LY294002, or the eNOS inhibitor, L-NAME, blocked the cardiovascular protective effects of fasudil. Conclusions-Inhibition of Rho-kinase leads to the activation of the PI3-kinase/Akt/eNOS pathway and cardiovascular protection. These findings suggest that Rho-kinase may play an important role in mediating the inflammatory response to I/R injury.

This study reports on what we believe are novel mechanism(s) of the vascular protective action of adiponectin. We used intravital microscopy to measure leukocyte-endothelium interactions in adiponectin-deficient (Ad -/-) mice and found that adiponectin deficiency was associated with a 2-fold increase in leukocyte rolling and a 5-fold increase in leukocyte adhesion in the microcirculation. Measurement of endothelial NO (eNO) revealed that adiponectin deficiency drastically reduced levels of eNO in the vascular wall. Immunohistochemistry demonstrated increased expression of E-selectin and VCAM-1 in the vascular endothelium of Ad -/-mice. Systemic administration of the recombinant globular adiponectin domain (gAd) to Ad -/-mice significantly attenuated leukocyte-endothelium interactions and adhesion molecule expression in addition to restoring physiologic levels of eNO. Importantly, prior administration of gAd also protected WT mice against TNF-α-induced leukocyte-endothelium interactions, indicating a pharmacologic action of gAd. Mechanistically, blockade of eNOS with N ω -nitro-l-arginine methyl ester (l-NAME) abolished the inhibitory effect of gAd on leukocyte adhesion, demonstrating the obligatory role of eNOS signaling in the antiinflammatory action of gAd. We believe this is the first demonstration that gAd protects the vasculature in vivo via increased NO bioavailability with suppression of leukocyte-endothelium interactions. Overall, we provide evidence that loss of adiponectin induces a primary state of endothelial dysfunction with increased leukocyte-endothelium adhesiveness.

Cardiovascular complications are the leading cause of morbidity and mortality in diabetic patients. Endothelial dysfunction with impaired endothelial nitric oxide (NO) synthase (eNOS) activity is a widely accepted cause of diabetic vasculopathy. The mechanisms of endothelial dysfunction in diabetes remain elusive, thus limiting effective therapeutic interventions. We report novel evidence demonstrating that the calciumdependent protease calpain causes endothelial dysfunction and vascular inflammation in the microcirculation of the ZDF (Zucker diabetic fatty) rat, a genetic rat model of type 2 diabetes. We found evidence of increased calpain activity and leukocyte trafficking in the microcirculation of ZDF rats. Inhibition of calpain activity significantly attenuated leukocyte-endothelium interactions in the vasculature of ZDF rats. Expression of cell adhesion molecules in the vascular endothelium of ZDF rats was consistently increased, and it was suppressed by calpain inhibition. In vivo measurement of endothelial NO availability demonstrated a 60% decrease in NO levels in the microcirculation of diabetic rats, which was also prevented by calpain inhibition. Immunoprecipitation studies revealed calpain-dependent loss of association between eNOS and the regulatory protein heat shock protein 90. Collectively, these data provide evidence for a novel mechanism of endothelial dysfunction and vascular inflammation in diabetes. Calpains may represent a new molecular target for the prevention and treatment of diabetic vascular complications. Diabetes 54:1132-1140, 2005 T he prevalence of type 2 diabetes is increasing dramatically in the U.S., with the number of patients diagnosed with type 2 diabetes escalating across all age-groups (1). Although vascular complications are the leading cause of morbidity and mortality in diabetic patients (1), the mechanisms responsible for diabetic vascular dysfunction remain poorly understood. Metabolic hallmarks of type 2 diabetes include hyperglycemia, obesity, and dyslipidemia, all of which are known risk factors for the development of vascular disease. All of these factors, especially hyperglycemia, have been associated with endothelial dysfunction (2). Overall, it is widely accepted that the endothelial dysfunction present in diabetic patients is a primary contributing factor to vascular complications.Several studies in both animals (3,4) and humans (5,6) have demonstrated that hyperglycemia/diabetes causes endothelial dysfunction characterized by a loss of endotheliumderived nitric oxide (NO) and increased oxidative stress. Endothelial NO is an important regulator of vascular homeostasis. Loss of NO results in endothelial dysfunction, leading to increased vascular tone and abnormal endothelial adhesiveness, which increases platelet aggregation and leukocyte trafficking at the vessel wall (7). Thus, the diabetic vasculature experiences increased oxidative stress and abnormal inflammatory signals (8). Among other approaches, the mechanisms responsible for the loss of NO in the hype...

Background:The rarity of neuroendocrine malignancies limits the ability to develop new therapies and thus a better understanding of the underlying biology is critical.Methods:Through a prospective, IRB-approved protocol, patients with neuroendocrine malignancies underwent next-generation sequencing of their tumours to detect somatic mutations (SMs) in 50 cancer-related genes. Clinicopathologic correlation was made among poorly differentiated neuroendocrine carcinomas (NECs/poorly differentiated histology and Ki-67 >20%) and pancreatic neuroendocrine tumours (PanNETs/Ki67 ⩽20%) and non-pancreatic neuroendocrine tumours (NP-NETs/Ki67 ⩽20%).Results:A total of 77 patients were enrolled, with next-generation sequencing results available on 63 patients. Incidence of SMs was 83% (19 out of 23) in poorly differentiated NECs, 45% (5 out of 11) in PanNETs and 14% (4 out of 29) in NP-NETs. TP53 was the most prevalent mutation in poorly differentiated NECs (57%), and KRAS (30%), PIK3CA/PTEN (22%) and BRAF (13%) mutations were also found. Small intestinal neuroendocrine tumours (Ki67 <2%/n=9) did not harbour any mutations. Prevalence of mutations correlated with higher risk of progression within the previous year (32% (low risk) vs 11% (high risk), P=0.01) and TP53 mutation correlated with worse survival (2-year survival 66% vs 97%, P=0.003).Conclusions:Poorly differentiated NECs have a high mutation burden with potentially targetable mutations. The TP53 mutations are associated with poor survival in neuroendocrine malignancies. These findings have clinical trial implications for choice of therapy and prognostic stratification and warrant confirmation.

The growth of disseminated tumor cells (DTCs) into metastatic lesions depends on the establishment of a favorable microenvironment in the stroma of the target organs. Here we show that mice treated with anakinra, an antagonist of the IL-1β receptor (IL-1R), or harboring a targeted deletion of IL-1R are significantly less prone to develop bone tumors when inoculated in the arterial circulation with human prostate cancer (PCa) cells expressing IL-1β. Interestingly, human mesenchymal stem cells (hMSCs) exposed in vitro to medium conditioned by IL-1β-expressing cancer cells responded by up regulating S100A4, a marker of cancer-associated fibroblasts (CAFs), and this effect was blocked by anakinra. Analogously, the stroma adjacent to skeletal metastases generated in mice by IL-1β-expressing cancer cells showed a dramatic increase in S100A4, COX-2 and the alteration of thirty tumor-related genes as measured by Nanostring analysis. These effects were not observed in the stroma associated to the rare and much smaller metastases generated by the same cells in IL-1R knockout animals, confirming that tumor-secreted IL-1β generates skeletal CAFs and conditions the surrounding bone microenvironment. In skeletal lesions from patients with metastatic PCa, histological and molecular analyses revealed that IL-1β is highly expressed in cancer cells in which the androgen receptor (AR) is not detected (AR−) whereas this cytokine is uniformly absent in the AR-positive (AR+) metastatic cells. The stroma conditioned by IL-1β-expressing cancer cells served as a supportive niche also for coexisting IL-1β-lacking cancer cells, which are otherwise unable to generate tumors after independently seeding the skeleton of mice. This niche is established very early following tumor seeding and hints to a role of IL-1β in promoting early colonization of PCa at the skeletal level.

Increased permeability to albumin is a well-known feature of diabetic microvasculature and a negative prognostic factor of vascular complications. The mechanisms responsible for loss of the physiological albumin barrier in diabetic organs remain only partially understood. We have recently demonstrated that the protease -calpain is activated in hyperglycemia, which causes endothelial dysfunction and vascular inflammation. In the present study, we investigated whether -calpain is involved in the hyperpermeability of the diabetic vasculature. We also investigated the mechanistic roles of hyperglycemia and leukocyte adhesion in this process. Albumin permeability in the intact microcirculation of the Zucker diabetic fatty (ZDF) rat was quantified by intravital microscopy. Extravasation of albumin in the microcirculation of ZDF rats was significantly increased when compared with nondiabetic Zucker lean (ZL) rats. Microvascular albumin leakage was prevented by either antisense depletion of -calpain or pharmacological inhibition of calpain in vivo. Calpain inhibition also attenuated urinary albumin excretion in ZDF rats. Glucose concentrations in the range of those found in the blood of ZDF rats increased albumin permeability in nondiabetic ZL rats. Thus, this demonstrates a mechanistic role for hyperglycemia in the hypermeability of diabetes. Depletion of polymorphonuclear leukocytes in vivo failed to prevent glucose-induced hypermeability, which suggests that hyperglycemia can disrupt the physiological endothelial cell barrier of the microcirculation, even in the absence of increased overt leukocyte-endothelium interactions.

Rationale The cytosolic protease calpain has been recently implicated in the vascular remodeling of angiotensin II (AngII) type-1 receptor (AT1r) signaling. The role of AngII/AT1r/calpain signaling on endothelial function, an important and early determinant of vascular pathology, remains though totally unknown. Accordingly, we investigated the role of calpain in the endothelial dysfunction of AngII. Objective To demonstrate a mechanistic role for calpain in the endothelial dysfunction induced by AngII/AT1r signaling. To establish endothelial-expressed calpains as an important target of AT1r signaling. Methods and Results Subchronic administration of nonpressor doses of AngII to rats and mice significantly increased vascular calpain activity via AT1r signaling. Intravital microscopy studies revealed that activation of vascular expressed calpains causes endothelial dysfunction with increased leukocyte-endothelium interactions and albumin permeability in the microcirculation. Western blot and immunohistochemistry studies confirmed that AngII/AT1r signaling preferentially activates the constitutively expressed μ-calpain isoform and demonstrated a calpain-dependent degradation of IκBα, along with upregulation of NF-κB-regulated endothelial cell adhesion molecules (eCAMs). These physiological and biochemical parameters were nearly normalized following inhibition of AT1r or calpain in vivo. Antisense depletion studies in microvascular endothelial cells along with knockout and transgenic mouse studies further confirmed the role of μ-calpain in the endothelial adhesiveness induced by AngII. Conclusions This study uncovers a novel role for calpain in the endothelial dysfunction of AngII/AT1r signaling and establishes the calpain system as a novel molecular target of the vascular protective action of RAS inhibition. Our results may have significant clinical implications in vascular disease.

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