Mitochondrial ATP production is required for endothelial cell control of vascular tone

Arteries and veins are lined by non-proliferating endothelial cells that play a critical role in regulating blood flow. Endothelial cells also regulate tissue perfusion, metabolite exchange, and thrombosis. It is thought that endothelial cells rely on ATP generated via glycolysis to fuel each of these energy-demanding processes. However, endothelial metabolism has mainly been studied in the context of proliferative cells in angiogenesis, and little is known about energy production in endothelial cells within the fully-formed vascular wall. Using intact arteries isolated from rats and mice, we show that inhibiting mitochondrial oxidative phosphorylation disrupts endothelial control of vascular tone. The role for endothelial cell energy production is independent of species, sex, or vascular bed. Basal, mechanically-activated, and agonist-evoked calcium activity in intact artery endothelial cells are each prevented by inhibiting mitochondrial ATP synthesis. This effect is mimicked by blocking the transport of pyruvate, the master fuel for mitochondrial energy production, through the mitochondrial pyruvate carrier. These data show that mitochondrial ATP is necessary for calcium-dependent, nitric oxide mediated endothelial control of vascular tone, and identifies the critical role of endothelial mitochondrial energy production in fueling perfused blood vessel function.

Inhibition of HDAC6 Activity Protects Against Endothelial Dysfunction and Atherogenesis in vivo: A Role for HDAC6 Neddylation

We previously reported that histone deacetylase 6 (HDAC6) has an important role in endothelial cell (EC) function in vitro. However, whether HDAC6 plays a role in atherogenesis in vivo and the mechanism(s) that control HDAC6 activity/expression in response to atherogenic stimuli are unclear. The goals of this study were to determine whether HDAC6 inhibitor tubacin attenuates atherogenesis and to elucidate specific molecular mechanism(s) that regulate endothelial HDAC6 expression/activity. We evaluated whether administration of tubacin attenuated or reversed the endothelial dysfunction and atherosclerosis induced in mice by a single intraperitoneal injection of adeno-associated viruses encoding liver-target PCSK9 gain-of-function mutant followed by a high fat diet (HFD) for 18 weeks. Tubacin significantly blunted PCSK9-induced increases in pulse wave velocity (index of vascular stiffness and overall vascular health) that are also seen in atherogenic mice. Furthermore, tubacin protected vessels from defective vasorelaxation, as evaluated by acetylcholine-mediated relaxation using wire myograph. Plaque burden defined by Oil Red O staining was also found to be significantly less in mice that received tubacin than in those that received PCSK9 alone. Inhibition of the NEDDylation pathway with MLN4924, an inhibitor of NEDD8-activating enzyme 1 (NAE1), significantly increased HDAC6 activity in HAECs. Interestingly, HDAC6 expression remained unchanged. Further, HAECs exposed to the atherogenic stimulus oxidized low-density lipoprotein (OxLDL) exhibited enhanced HDAC6 activity, which was attenuated by pretreatment with MLN4924. The HDAC6 NEDDylation molecular pathway might regulate genes related to endothelial control of vasomotor tone, reactivity, and atherosclerosis. Tubacin may represent a novel pharmacologic intervention for atherogenesis and other vasculopathies.

Increased Vascular Contractility in Hypertension Results From Impaired Endothelial Calcium Signaling

Endothelial cells line all blood vessels and are critical regulators of vascular tone. In hypertension, disruption of endothelial function alters the release of endothelial-derived vasoactive factors and results in increased vascular tone. Although the release of endothelial-derived vasodilators occurs in a Ca 2+ -dependent manner, little is known on how Ca 2+ signaling is altered in hypertension. A key element to endothelial control of vascular tone is Ca 2+ signals at specialized regions (myoendothelial projections) that connect endothelial cells and smooth muscle cells. This work describes disruption in the operation of this key Ca 2+ signaling pathway in hypertension. We show that vascular reactivity to phenylephrine is increased in hypertensive (spontaneously hypertensive rat) when compared with normotensive (Wistar Kyoto) rats. Basal endothelial Ca 2+ activity limits vascular contraction, but that Ca 2+ -dependent control is impaired in hypertension. When changes in endothelial Ca 2+ levels are buffered, vascular contraction to phenylephrine increased, resulting in similar responses in normotension and hypertension. Local endothelial IP 3 (inositol trisphosphate)-mediated Ca 2+ signals are smaller in amplitude, shorter in duration, occur less frequently, and arise from fewer sites in hypertension. Spatial control of endothelial Ca 2+ signaling is also disrupted in hypertension: local Ca 2+ signals occur further from myoendothelial projections in hypertension. The results demonstrate that the organization of local Ca 2+ signaling circuits occurring at myoendothelial projections is disrupted in hypertension, giving rise to increased contractile responses.

The role of small and intermediate conductance calcium-activated potassium channels in endothelial-dependent hyperpolarization in physiology and arterial hypertension

The endothelium plays a crucial role in modulating vascular tone by synthesizing and releasing endothelium-derived relaxing factors, including nitric oxide (NO) and prostacyclin I2 (PGI2). Additionally, endothelium-dependent hyperpolarization (EDH) that is NO – and PGI2–independent participates in the relaxation of small-diameter blood vessels (<300 μm). EDH response is initiated by agonists (e.g. acetylcholine, bradykinin) – or shear stress – induced increase of calcium ions level in the endothelium and involves opening of the endothelial small (KCa2.3) and intermediate conductance (KCa3.1) calcium-activated potassium channels. The efflux of potassium ions could elicit the hyperpolarization of the surrounding myocytes by the activation of the inward-rectifier potassium ion channel (KIR) and/or Na+/K+-ATPase. The reduced release and/or bioavailability of NO, which is characteristic for endothelial dysfunction and may result in arterial hypertension, stimulate the generation of EDH signals, as a compensatory mechanism to maintain the endothelial control of vasodilator tone. The contribution of EDH in endothelium-dependent relaxation varies between vascular beds, animal and experimental model. In arterial hypertension the reduced expression/activity of KCa3.1 and KCa2.3 results in impaired vasorelaxation. Currently, the use of modulatory compounds (activators and inhibitors) of KCa3.1 and KCa2.3 as the potential therapeutic targets in cardiovascular diseases is under intensive investigation. It has already been known that application of activators of KCa3.1 and KCa2.3 potassium channels such (as SKA-31) can improve the EDH-type responses, the endothelial function and decrease mean arterial blood pressure. This may suggest the usefulness of these compounds in the treatment of arterial hypertension.

NIDOGEN1 as a novel regulator of endothelial control over breast cancer invasion and metastasis.

e23005 Background: The microenvironment is a central regulator of cancer biology. While the contribution of fibroblasts has been largely studied, the role of endothelial cells as regulators of cancer cell behavior is still poorly understood. As in a diverse spectrum of physiological processes in normal tissue, endothelial cells may exert a similar regulatory control in cancer progression and metastasis. Methods: To characterize the functional effects of endothelial-cancer interaction we focused on an in vitro co-culture model. Results: Co-culturing human umbilical venous endothelial cells (HUVEC) with SKBR-3 breast cancer cells induced morphological changes with epithelial-mesenchymal transition traits (EMT) and a significantly increased migratory and invasive potential. This activity leading to an elongated phenotype, expression of mesenchymal markers and pro-migratory gene sets in SKBR-3 was contained in HUVEC conditioned medium. The pro-migratory effect on SKBR-3 was significantly more pronounced when the supernatant was obtained from a sub-confluent and highly proliferative endothelial cell culture than from a confluent and resting endothelial cell layer. To identify the secreted regulatory molecules, we analyzed the supernatant of sub-confluent and confluent endothelial cells by quantitative MS proteomics (SILAC analysis). Eight candidate proteins significantly more secreted in conditioned medium from confluent HUVEC represented potential inhibitors of migration. Among them NIDOGEN1 was found to be necessary and sufficient for the inhibition of EMT and migration in SKBR-3. Stimulation of SKBR-3 with supernatant from sub-confluent HUVEC increased p-STAT3 levels in SKBR-3. Silencing nidogen1 in confluent HUVEC re-activated phosphorylation of STAT3 indicating that NIDOGEN1 inhibits the promigratory STAT3 pathway. The STAT3 pathway and migration were also inhibited by overexpression of nidogen1 in MDA-MB-231 LM2 cells.When injected in the mammary fat pad of nude mice these cells formed significantly less lung metastases than controls (p < 0.01). Conclusions: We identified NIDOGEN1 as a novel regulator of endothelial control over cancer cell migration, invasion and metastasis.