Victor Blanco

Tone-dependent vascular responses to astrocyte-derived signals.

A growing number of studies support an important contribution of astrocytes to neurovascular coupling, i.e., the phenomenon by which variations in neuronal activity trigger localized changes in blood flow that serve to match the metabolic demands of neurons. However, since both constriction and dilations have been observed in brain parenchymal arterioles upon astrocyte stimulation, the specific influences of these cells on the vasculature remain unclear. Using acute brain slices, we present evidence showing that the specific degree of constriction of rat cortical arterioles (vascular tone) is a key determinant of the magnitude and polarity of the diameter changes elicited by signals associated with neurovascular coupling. Thus elevation of extracellular K+ concentration, stimulation of metabotropic glutamate receptors (mGluR), or 11,12-epoxyeicosatrienoic acid application all elicited vascular responses that were affected by the particular resting arteriolar tone. Interestingly, the data suggest that the extent and/or polarity of the vascular responses are influenced by a delimited set point centered between 30 and 40% tone. In addition, we report that distinct, tone-dependent effects on arteriolar diameter occur upon stimulation of mGluR during inhibition of enzymes of the arachidonic acid pathway [i.e., phospholipase A2, cytochrome P-450 (CYP) omega-hydroxylase, CYP epoxygenase, and cycloxygenase-1]. Our findings may reconcile previous evidence in which direct astrocytic stimulation elicited either vasoconstrictions or vasodilations and also suggest the novel concept that, in addition to participating in functional hyperemia, astrocyte-derived signals play a role in adjusting vascular tone to a range where dilator responses are optimal.

Neurovascular coupling in the mammalian brain

Normal brain function requires proper supply of oxygen and glucose in a timely and local manner. This is achieved through an orchestrated intercellular communication between neurones, astrocytes and microvessels that results in a rapid and restricted increase in cerebral blood flow, a process known as neurovascular coupling. Astrocytic end‐feet make close contacts with neuronal synapses and blood vessels and, given their ability to release vasoactive signals following neuronal activation, have been recognized as key intermediaries in the neurovascular response. Both dilating and constricting signals appear to be released from astrocytes upon increases in intracellular Ca2+ concentration, and both dilatation and constriction of brain vessels have been observed in previous studies. In this article, we discuss the various astrocyte‐derived vasodilating and vasoconstricting signals, their interactions and effects on astrocytes and vascular smooth muscle cells, and suggest the importance of the intrinsic properties of the latter cell type on the overall neurovascular response. We present a working model in which the rise in astrocytic Ca2+ following neuronal activation leads not only to the rapid activation of calcium‐activated K+ channels in astrocytic end‐feet, but also to their modulation by metabolites of the arachidonic acid pathway, which in general have been proposed to act on vascular smooth muscle cells rather than on astrocytes. We propose that this latter mechanism may in turn modulate K+ signalling from astrocytes to smooth muscle cells, influencing the overall effects of the vasodilating and vasoconstricting signals released during neuronal activation.

Histone Deacetylase 9 Is a Negative Regulator of Adipogenic Differentiation

Differentiation of preadipocytes into mature adipocytes capable of efficiently storing lipids is an important regulatory mechanism in obesity. Here, we examined the involvement of histone deacetylases (HDACs) and histone acetyltransferases (HATs) in the regulation of adipogenesis. We find that among the various members of the HDAC and HAT families, only HDAC9 exhibited dramatic down-regulation preceding adipogenic differentiation. Preadipocytes from HDAC9 gene knock-out mice exhibited accelerated adipogenic differentiation, whereas HDAC9 overexpression in 3T3-L1 preadipocytes suppressed adipogenic differentiation, demonstrating its direct role as a negative regulator of adipogenesis. HDAC9 expression was higher in visceral as compared with subcutaneous preadipocytes, negatively correlating with their potential to undergo adipogenic differentiation in vitro. HDAC9 localized in the nucleus, and its negative regulation of adipogenesis segregates with the N-terminal nuclear targeting domain, whereas the C-terminal deacetylase domain is dispensable for this function. HDAC9 co-precipitates with USF1 and is recruited with USF1 at the E-box region of the C/EBPα gene promoter in preadipocytes. Upon induction of adipogenic differentiation, HDAC9 is down-regulated, leading to its dissociation from the USF1 complex, whereas p300 HAT is up-regulated to allow its association with USF1 and accumulation at the E-box site of the C/EBPα promoter in differentiated adipocytes. This reciprocal regulation of HDAC9 and p300 HAT in the USF1 complex is associated with increased C/EBPα expression, a master regulator of adipogenic differentiation. These findings provide new insights into mechanisms of adipogenic differentiation and document a critical regulatory role for HDAC9 in adipogenic differentiation through a deacetylase-independent mechanism.

Role of epoxyeicosatrienoic acids as autocrine metabolites in glutamate-mediated K+ signaling in perivascular astrocytes

Epoxyeicosatrienoic acids (EETs), synthesized and released by astrocytes in response to glutamate, are known to play a pivotal role in neurovascular coupling. In vascular smooth muscle cells (VSMC), EETs activate large-conductance, Ca(2+)-activated K(+) (BK) channels resulting in hyperpolarization and vasodilation. However, the functional role and mechanism of action for glial-derived EETs are still to be determined. In this study, we evaluated the effect of the synthetic EET analog 11-nonyloxy-undec-8(Z)-enoic acid (NUD-GA) on outward K(+) currents mediated by calcium-activated K(+) channels. Addition of NUD-GA significantly increased intracellular Ca(2+) and outward K(+) currents in perivascular astrocytes. NUD-GA-induced currents were significantly inhibited by BK channel blockers paxilline and tetraethylammonium (TEA) (23.4 ± 2.4%; P < 0.0005). Similarly, NUD-GA-induced currents were also significantly inhibited in the presence of the small-conductance Ca(2+)-activated K(+) channel inhibitor apamin along with a combination of blockers against glutamate receptors (12.8 ± 2.70%; P < 0.05). No changes in outward currents were observed in the presence of the channel blocker for intermediate-conductance K(+) channels TRAM-34. Blockade of the endogenous production of EETs with N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH) significantly blunted (dl)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD)-induced outward K(+) currents (P < 0.05; n = 6). Both NUD-GA and t-ACPD significantly increased BK channel single open probability; the later was blocked following MS-PPOH incubation. Our data supports the idea that EETs are potent K(+) channel modulators in cortical perivascular astrocytes and further suggest that these metabolites may participate in NVC by modulating the levels of K(+) released at the gliovascular space.

HDAC9 Knockout Mice Are Protected From Adipose Tissue Dysfunction and Systemic Metabolic Disease During High-Fat Feeding

During chronic caloric excess, adipose tissue expands primarily by enlargement of individual adipocytes, which become stressed with lipid overloading, thereby contributing to obesity-related disease. Although adipose tissue contains numerous preadipocytes, differentiation into functionally competent adipocytes is insufficient to accommodate the chronic caloric excess and prevent adipocyte overloading. We report for the first time that a chronic high-fat diet (HFD) impairs adipogenic differentiation, leading to accumulation of inefficiently differentiated adipocytes with blunted expression of adipogenic differentiation-specific genes. Preadipocytes from these mice likewise exhibit impaired adipogenic differentiation, and this phenotype persists during in vitro cell culture. HFD-induced impaired adipogenic differentiation is associated with elevated expression of histone deacetylase 9 (HDAC9), an endogenous negative regulator of adipogenic differentiation. Genetic ablation of HDAC9 improves adipogenic differentiation and systemic metabolic state during an HFD, resulting in diminished weight gain, improved glucose tolerance and insulin sensitivity, and reduced hepatosteatosis. Moreover, compared with wild-type mice, HDAC9 knockout mice exhibit upregulated expression of beige adipocyte marker genes, particularly during an HFD, in association with increased energy expenditure and adaptive thermogenesis. These results suggest that targeting HDAC9 may be an effective strategy for combating obesity-related metabolic disease.

Astrocyte Contributions to Flow/Pressure-Evoked Parenchymal Arteriole Vasoconstriction

Basal and activity-dependent cerebral blood flow changes are coordinated by the action of critical processes, including cerebral autoregulation, endothelial-mediated signaling, and neurovascular coupling. The goal of our study was to determine whether astrocytes contribute to the regulation of parenchymal arteriole (PA) tone in response to hemodynamic stimuli (pressure/flow). Cortical PA vascular responses and astrocytic Ca2+ dynamics were measured using an in vitro rat/mouse brain slice model of perfused/pressurized PAs; studies were supplemented with in vivo astrocytic Ca2+ imaging. In vitro, astrocytes responded to PA flow/pressure increases with an increase in intracellular Ca2+. Astrocytic Ca2+ responses were corroborated in vivo, where acute systemic phenylephrine-induced increases in blood pressure evoked a significant increase in astrocytic Ca2+. In vitro, flow/pressure-evoked vasoconstriction was blunted when the astrocytic syncytium was loaded with BAPTA (chelating intracellular Ca2+) and enhanced when high Ca2+ or ATP were introduced to the astrocytic syncytium. Bath application of either the TRPV4 channel blocker HC067047 or purinergic receptor antagonist suramin blunted flow/pressure-evoked vasoconstriction, whereas K+ and 20-HETE signaling blockade showed no effect. Importantly, we found TRPV4 channel expression to be restricted to astrocytes and not the endothelium of PA. We present evidence for a novel role of astrocytes in PA flow/pressure-evoked vasoconstriction. Our data suggest that astrocytic TRPV4 channels are key molecular sensors of hemodynamic stimuli and that a purinergic, glial-derived signal contributes to flow/pressure-induced adjustments in PA tone. Together our results support bidirectional signaling within the neurovascular unit and astrocytes as key modulators of PA tone.

CD14 directs adventitial macrophage precursor recruitment: role in early abdominal aortic aneurysm formation.

Background Recruitment of macrophage precursors to the adventitia plays a key role in the pathogenesis of abdominal aortic aneurysms (AAAs), but molecular mechanisms remain undefined. The innate immune signaling molecule CD14 was reported to be upregulated in adventitial macrophages in a murine model of AAA and in monocytes cocultured with aortic adventitial fibroblasts (AoAf) in vitro, concurrent with increased interleukin‐6 (IL‐6) expression. We hypothesized that CD14 plays a crucial role in adventitial macrophage precursor recruitment early during AAA formation. Methods and Results CD14−/− mice were resistant to AAA formation induced by 2 different AAA induction models: aortic elastase infusion and systemic angiotensin II (AngII) infusion. CD14 gene deletion led to reduced aortic macrophage infiltration and diminished elastin degradation. Adventitial monocyte binding to AngII‐infused aorta in vitro was dependent on CD14, and incubation of human acute monocytic leukemia cell line‐1 (THP‐1) monocytes with IL‐6 or conditioned medium from perivascular adipose tissue (PVAT) upregulated CD14 expression. Conditioned medium from AoAf and PVAT induced CD14‐dependent monocyte chemotaxis, which was potentiated by IL‐6. CD14 expression in aorta and plasma CD14 levels were increased in AAA patients compared with controls. Conclusions These findings link CD14 innate immune signaling via a novel IL‐6 amplification loop to adventitial macrophage precursor recruitment in the pathogenesis of AAA.

Variation in human cancer cell external phosphatidylserine is regulated by flippase activity and intracellular calcium

Viable cancer cells expose elevated levels of phosphatidylserine (PS) on the exoplasmic face of the plasma membrane. However, the mechanisms leading to elevated PS exposure in viable cancer cells have not been defined. We previously showed that externalized PS may be used to monitor, target and kill tumor cells. In addition, PS on tumor cells is recognized by macrophages and has implications in antitumor immunity. Therefore, it is important to understand the molecular details of PS exposure on cancer cells in order to improve therapeutic targeting. Here we explored the mechanisms regulating the surface PS exposure in human cancer cells and found that differential flippase activity and intracellular calcium are the major regulators of surface PS exposure in viable human cancer cells. In general, cancer cell lines with high surface PS exhibited low flippase activity and high intracellular calcium, whereas cancer cells with low surface PS exhibited high flippase activity and low intracellular calcium. High surface PS cancer cells also had higher total cellular PS than low surface PS cells. Together, our results indicate that the amount of external PS in cancer cells is regulated by calcium dependent flippase activity and may also be influenced by total cellular PS.

Red Blood Cell Dysfunction Induced by High-Fat Diet: Potential Implications for Obesity-Related Atherosclerosis

Background— High-fat diet (HFD) promotes endothelial dysfunction and proinflammatory monocyte activation, which contribute to atherosclerosis in obesity. We investigated whether HFD also induces the dysfunction of red blood cells (RBCs), which serve as a reservoir for chemokines via binding to Duffy antigen receptor for chemokines (DARC). Methods and Results— A 60% HFD for 12 weeks, which produced only minor changes in lipid profile in C57/BL6 mice, markedly augmented the levels of monocyte chemoattractant protein-1 bound to RBCs, which in turn stimulated macrophage migration through an endothelial monolayer. Levels of RBC-bound KC were also increased by HFD. These effects of HFD were abolished in DARC–/– mice. In RBCs from HFD-fed wild-type and DARC–/– mice, levels of membrane cholesterol and phosphatidylserine externalization were increased, fostering RBC-macrophage inflammatory interactions and promoting macrophage phagocytosis in vitro. When labeled ex vivo and injected into wild-type mice, RBCs from HFD-fed mice exhibited ≈3-fold increase in splenic uptake. Finally, RBCs from HFD-fed mice induced increased macrophage adhesion to the endothelium when they were incubated with isolated aortic segments, indicating endothelial activation. Conclusions— RBC dysfunction, analogous to endothelial dysfunction, occurs early during diet-induced obesity and may serve as a mediator of atherosclerosis. These findings may have implications for the pathogenesis of atherosclerosis in obesity, a worldwide epidemic. # CLINICAL PERSPECTIVE {#article-title-42}Background— High-fat diet (HFD) promotes endothelial dysfunction and proinflammatory monocyte activation, which contribute to atherosclerosis in obesity. We investigated whether HFD also induces the dysfunction of red blood cells (RBCs), which serve as a reservoir for chemokines via binding to Duffy antigen receptor for chemokines (DARC). Methods and Results— A 60% HFD for 12 weeks, which produced only minor changes in lipid profile in C57/BL6 mice, markedly augmented the levels of monocyte chemoattractant protein-1 bound to RBCs, which in turn stimulated macrophage migration through an endothelial monolayer. Levels of RBC-bound KC were also increased by HFD. These effects of HFD were abolished in DARC–/– mice. In RBCs from HFD-fed wild-type and DARC–/– mice, levels of membrane cholesterol and phosphatidylserine externalization were increased, fostering RBC-macrophage inflammatory interactions and promoting macrophage phagocytosis in vitro. When labeled ex vivo and injected into wild-type mice, RBCs from HFD-fed mice exhibited ≈3-fold increase in splenic uptake. Finally, RBCs from HFD-fed mice induced increased macrophage adhesion to the endothelium when they were incubated with isolated aortic segments, indicating endothelial activation. Conclusions— RBC dysfunction, analogous to endothelial dysfunction, occurs early during diet-induced obesity and may serve as a mediator of atherosclerosis. These findings may have implications for the pathogenesis of atherosclerosis in obesity, a worldwide epidemic.

SapC–DOPS Nanovesicles as Targeted Therapy for Lung Cancer

Lung cancer is the deadliest type of cancer for both men and women. In this study, we evaluate the in vitro and in vivo efficacy of a biotherapeutic agent composed of a lysosomal protein (Saposin C, SapC) and a phospholipid (dioleoylphosphatidylserine, DOPS), which can be assembled into nanovesicles (SapC–DOPS) with selective antitumor activity. SapC–DOPS targets phosphatidylserine, an anionic phospholipid preferentially exposed in the surface of cancer cells and tumor-associated vasculature. Because binding of SapC to phosphatidylserine is favored at acidic pHs, and the latter characterizes the milieu of many solid tumors, we tested the effect of pH on the binding capacity of SapC–DOPS to lung tumor cells. Results showed that SapC–DOPS binding to cancer cells was more pronounced at low pH. Viability assays on a panel of human lung tumor cells showed that SapC–DOPS cytotoxicity was positively correlated with cell surface phosphatidylserine levels, whereas mitochondrial membrane potential measurements were consistent with apoptosis-related cell death. Using a fluorescence tracking method in live mice, we show that SapC–DOPS specifically targets human lung cancer xenografts, and that systemic therapy with SapC–DOPS induces tumor apoptosis and significantly inhibits tumor growth. These results suggest that SapC–DOPS nanovesicles are a promising treatment option for lung cancer. Mol Cancer Ther; 14(2); 491–8. ©2015 AACR.