Caveolae in endothelial cells have been implicated as plasma membrane microdomains that sense or transduce hemodynamic changes into biochemical signals that regulate vascular function. Therefore we compared long- and short-term flow-mediated mechanotransduction in vessels from WT mice, caveolin-1 knockout (Cav-1 KO) mice, and Cav-1 KO mice reconstituted with a transgene expressing Cav-1 specifically in endothelial cells (Cav-1 RC mice). Arterial remodeling during chronic changes in flow and shear stress were initially examined in these mice. Ligation of the left external carotid for 14 days to lower blood flow in the common carotid artery reduced the lumen diameter of carotid arteries from WT and Cav-1 RC mice. In Cav-1 KO mice, the decrease in blood flow did not reduce the lumen diameter but paradoxically increased wall thickness and cellular proliferation. In addition, in isolated pressurized carotid arteries, flow-mediated dilation was markedly reduced in Cav-1 KO arteries compared with those of WT mice. This impairment in response to flow was rescued by reconstituting Cav-1 into the endothelium. In conclusion, these results showed that endothelial Cav-1 and caveolae are necessary for both rapid and long-term mechanotransduction in intact blood vessels.

The genetic loss of endothelial-derived nitric oxide synthase (eNOS) in mice impairs vascular endothelial growth factor (VEGF) and ischemia-initiated blood flow recovery resulting in critical limb ischemia. This result may occur through impaired arteriogenesis, angiogenesis, or mobilization of stem and progenitor cells. Here, we show that after ischemic challenge, eNOS knockout mice [eNOS (-/-)] have defects in arteriogenesis and functional blood flow reserve after muscle stimulation and pericyte recruitment, but no impairment in endothelial progenitor cell recruitment. More importantly, the defects in blood flow recovery, clinical manifestations of ischemia, ischemic reserve capacity, and pericyte recruitment into the growing neovasculature can be rescued by local intramuscular delivery of an adenovirus encoding a constitutively active allele of eNOS, eNOS S1179D, but not a control virus. Collectively, our data suggest that endogenous eNOS-derived NO exerts direct effects in preserving blood flow, thereby promoting arteriogenesis, angiogenesis, and mural cell recruitment to immature angiogenic sprouts.

Caveolin-1 (Cav-1) is the principal structural component of caveolae organelles in smooth muscle cells, adipocytes, fibroblasts, epithelial cells, and endothelial cells (ECs). Cav-1–deficient (Cav-1 knockout [KO]) mice are viable and show increases of nitric oxide (NO) production in vasculature, cardiomyopathy, and pulmonary dysfunction. In this study, we generated EC-specific Cav-1–reconstituted (Cav-1 RC) mice and reexamined vascular, cardiac, and pulmonary phenotypes. Cav-1 KO pulmonary arteries had decreased smooth muscle contractility and increased endothelial NO synthase activation and hypotension; the latter two effects were rescued completely in Cav-1 RC mice. Cav-1 KO mice exhibited myocardial hypertrophy, pulmonary hypertension, and alveolar cell hyperproliferation caused by constitutive activation of p42/44 mitogen-activated protein kinase and Akt. Interestingly, in Cav-1 RC mice, cardiac hypertrophy and pulmonary hypertension were completely rescued, whereas alveolar hyperplasia was partially recovered because of the lack of rescue of Cav-1 in bronchiolar epithelial cells. These results provide clear physiological evidence supporting the important role of cell type–specific Cav-1 expression governing multiple phenotypes in the vasculature, heart, and lung.

Histamine is a mediator of allergic inflammation released mainly from mast cells. Although histamine strongly increases vascular permeability, its precise mechanism under in vivo situation remains unknown. We here attempted to reveal how histamine induces vascular hyperpermeability focusing on the key regulators of vascular permeability, blood flow and endothelial barrier. Degranulation of mast cells by antigen-stimulation or histamine treatment induced vascular hyperpermeability and tissue swelling in mouse ears. These were abolished by histamine H1 receptor antagonism. Intravital imaging showed that histamine dilated vasculature, increased blood flow, while it induced hyperpermeability in venula. Whole-mount staining showed that histamine disrupted endothelial barrier formation of venula indicated by changes in vascular endothelial cadherin (VE-cadherin) localization at endothelial cell junction. Inhibition of nitric oxide synthesis (NOS) by L-NAME or vasoconstriction by phenylephrine strongly inhibited the histamine-induced blood flow increase and hyperpermeability without changing the VE-cadherin localization. In vitro, measurements of trans-endothelial electrical resistance of human dermal microvascular endothelial cells (HDMECs) showed that histamine disrupted endothelial barrier. Inhibition of protein kinase C (PKC) or Rho-associated protein kinase (ROCK), NOS attenuated the histamine-induced barrier disruption. These observations suggested that histamine increases vascular permeability mainly by nitric oxide (NO)-dependent vascular dilation and subsequent blood flow increase and maybe partially by PKC/ROCK/NO-dependent endothelial barrier disruption.

Objective—: TGR5 is a G-protein–coupled receptor for bile acids. So far, little is known about the function of TGR5 in vascular endothelial cells. Approach and Results—: In bovine aortic endothelial cells, treatment with a bile acid having a high affinity to TGR5, taurolithocholic acid (TLCA), significantly increased NO production. This effect was abolished by small interfering RNA–mediated depletion of TGR5. TLCA-induced NO production was also observed in human umbilical vein endothelial cells measured via intracellular cGMP accumulation. TLCA increased endothelial NO synthase ^ser1177 phosphorylation in human umbilical vein endothelial cells. This response was accompanied by increased Akt ^ser473 phosphorylation and intracellular Ca ²⁺ . Inhibition of these signals significantly decreased TLCA-induced NO production. We next examined whether TGR5-mediated NO production affects inflammatory responses of endothelial cells. In human umbilical vein endothelial cells, TLCA significantly reduced tumor necrosis factor-α–induced adhesion of monocytes, vascular cell adhesion molecule-1 expression, and activation of nuclear factor-κB. TLCA also inhibited lipopolysaccharide-induced monocyte adhesion to mesenteric venules in vivo. These inhibitory effects of TLCA were abrogated by NO synthase inhibition. Conclusions—: TGR5 agonism induces NO production via Akt activation and intracellular Ca ²⁺ increase in vascular endothelial cells, and this function inhibits monocyte adhesion in response to inflammatory stimuli.