Comparative evaluation of trypsin and elastase digestion techniques for isolation of murine retinal vasculature.

Dysfunctional pericytes and disruption of adherens or tight junctions are related to many microvascular diseases, including diabetic retinopathy. In this context, visualizing retinal vascular architecture becomes essential for understanding retinal vascular disease pathophysiology. Although flat mounts provide a demonstration of the retinal blood vasculature, they often lack a clear view of microaneurysms and capillary architecture. Trypsin and elastase digestion are the two techniques for isolating retinal vasculatures in rats, mice, and other animal models. Our observations in the present study reveal that trypsin digestion impacts the association between pericytes and endothelial cells. In contrast, elastase digestion effectively preserves these features in the blood vessels. Furthermore, trypsin digestion disrupts endothelial adherens and tight junctions that elastase digestion does not. Therefore, elastase digestion emerges as a superior technique for isolating retinal vessels, which can be utilized to collect reliable and consistent data to comprehend the pathophysiology of disorders involving microvascular structures.

Vascular cell-adhesion molecule 1 (VCAM-1) regulates JunB-mediated IL-8/CXCL1 expression and pathological neovascularization.

Vascular adhesion molecules play an important role in various immunological disorders, particularly in cancers. However, little is known regarding the role of these adhesion molecules in proliferative retinopathies. We observed that IL-33 regulates VCAM-1 expression in human retinal endothelial cells and that genetic deletion of IL-33 reduces hypoxia-induced VCAM-1 expression and retinal neovascularization in C57BL/6 mice. We found that VCAM-1 via JunB regulates IL-8 promoter activity and expression in human retinal endothelial cells. In addition, our study outlines the regulatory role of VCAM-1-JunB-IL-8 signaling on retinal endothelial cell sprouting and angiogenesis. Our RNA sequencing results show an induced expression of CXCL1 (a murine functional homolog of IL-8) in the hypoxic retina, and intravitreal injection of VCAM-1 siRNA not only decreases hypoxia-induced VCAM-1-JunB-CXCL1 signaling but also reduces OIR-induced sprouting and retinal neovascularization. These findings suggest that VCAM-1-JunB-IL-8 signaling plays a crucial role in retinal neovascularization, and its antagonism might provide an advanced treatment option for proliferative retinopathies.

IL-33 via PKCµ/PRKD1 Mediated α-Catenin Phosphorylation Regulates Endothelial Cell-Barrier Integrity and Ischemia-Induced Vascular Leakage.

Angiogenesis, neovascularization, and vascular remodeling are highly dynamic processes, where endothelial cell-cell adhesion within the vessel wall controls a range of physiological processes, such as growth, integrity, and barrier function. The cadherin-catenin adhesion complex is a key contributor to inner blood-retinal barrier (iBRB) integrity and dynamic cell movements. However, the pre-eminent role of cadherins and their associated catenins in iBRB structure and function is not fully understood. Using a murine model of oxygen-induced retinopathy (OIR) and human retinal microvascular endothelial cells (HRMVECs), we try to understand the significance of IL-33 on retinal endothelial barrier disruption, leading to abnormal angiogenesis and enhanced vascular permeability. Using electric cell-substrate impedance sensing (ECIS) analysis and FITC-dextran permeability assay, we observed that IL-33 at a 20 ng/mL concentration induced endothelial-barrier disruption in HRMVECs. The adherens junction (AJs) proteins play a prominent role in the selective diffusion of molecules from the blood to the retina and in maintaining retinal homeostasis. Therefore, we looked for the involvement of adherens junction proteins in IL-33-mediated endothelial dysfunction. We observed that IL-33 induces α-catenin phosphorylation at serine/threonine (Ser/Thr) residues in HRMVECs. Furthermore, mass-spectroscopy (MS) analysis revealed that IL-33 induces the phosphorylation of α-catenin at Thr654 residue in HRMVECs. We also observed that PKCµ/PRKD1-p38 MAPK signaling regulates IL-33-induced α-catenin phosphorylation and retinal endothelial cell-barrier integrity. Our OIR studies revealed that genetic deletion of IL-33 resulted in reduced vascular leakage in the hypoxic retina. We also observed that the genetic deletion of IL-33 reduced OIR-induced PKCµ/PRKD1-p38 MAPK-α-catenin signaling in the hypoxic retina. Therefore, we conclude that IL-33-induced PKCµ/PRKD1-p38 MAPK-α-catenin signaling plays a significant role in endothelial permeability and iBRB integrity.

IL-33 enhances Jagged1 mediated NOTCH1 intracellular domain (NICD) deubiquitination and pathological angiogenesis in proliferative retinopathy.

Pathological retinal neovascularization (NV) is a clinical manifestation of various proliferative retinopathies, and treatment of NV using anti-VEGF therapies is not selective, as it also impairs normal retinal vascular growth and function. Here, we show that genetic deletion or siRNA-mediated downregulation of IL-33 reduces pathological NV in a murine model of oxygen-induced retinopathy (OIR) with no effect on the normal retinal repair. Furthermore, our fluorescent activated cell sorting (FACS) data reveals that the increase in IL-33 expression is in endothelial cells (ECs) of the hypoxic retina and conditional genetic deletion of IL-33 in retinal ECs reduces pathological NV. In vitro studies using human retinal microvascular endothelial cells (HRMVECs) show that IL-33 induces sprouting angiogenesis and requires NFkappaB-mediated Jagged1 expression and Notch1 activation. Our data also suggest that IL-33 enhances de-ubiquitination and stabilization of Notch1 intracellular domain via its interaction with BRCA1-associated protein 1 (BAP1) and Numb in HRMVECs and a murine model of OIR.

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol.

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of methods. Here, we describe two important approaches used for this purpose. First, we describe how to enrich tissues and cells with cholesterol using cyclodextrin saturated with cholesterol using cerebral arteries (tissues) and hippocampal neurons (cells) as examples. This approach can be used for any type of tissue, cells, or cell lines. An alternative approach for cholesterol enrichment involves the use of low-density lipoprotein (LDL). The advantage of this approach is that it uses part of the natural cholesterol homeostasis machinery of the cell. However, whereas the cyclodextrin approach can be applied to enrich any cell type of interest with cholesterol, the LDL approach is limited to cells that express LDL receptors (e.g., liver cells, bone marrow-derived cells such as blood leukocytes and tissue macrophages), and the level of enrichment depends on the concentration and the mobility of the LDL receptor. Furthermore, LDL particles include other lipids, so cholesterol delivery is nonspecific. Second, we describe how to enrich Xenopus oocytes with cholesterol using a phospholipid-based dispersion (i.e., liposomes) that includes cholesterol. Xenopus oocytes constitute a popular heterologous expression system used for studying cell and protein function. For both the cyclodextrin-based cholesterol enrichment approach of mammalian tissue (cerebral arteries) and for the phospholipid-based cholesterol enrichment approach of Xenopus oocytes, we demonstrate that cholesterol levels reach a maximum following 5 min of incubation. This level of cholesterol remains constant during extended periods of incubation (e.g., 60 min). Together, these data provide the basis for optimized temporal conditions for cholesterol enrichment of tissues, cells, and Xenopus oocytes for functional studies aimed at interrogating the impact of cholesterol enrichment.

Temporal Requirement for the Protective Effect of Dietary Cholesterol against Alcohol-Induced Vasoconstriction.

Moderate-to-heavy episodic alcohol drinking resulting in 30-80 mM of ethanol in blood constricts cerebral arteries and constitutes a risk factor for cerebrovascular disease. Alcohol-induced constriction of cerebral arteries in vivo and ex vivo has been shown to be blunted by dietary cholesterol (CLR) in a rat model of a high-CLR diet. Such protection has been proposed to arise from the high-CLR diet-driven increase in blood CLR levels and accompanying buildup of CLR within the cerebral artery smooth muscle. Here we used a rat model of high-CLR feeding in vivo and pressurized cerebral arteries ex vivo to examine whether the degree and time-course of alcohol-induced constriction are related to blood CLR levels. We demonstrate that subjecting young (3 weeks-old, 50 g) male Sprague-Dawley rats to a high- CLR feeding up to 41 weeks, resulted in an age-dependent increase in total blood CLR levels, when compared to those of age-matched rats on isocaloric (control) chow. This increase was paralleled by a high-CLR diet-driven elevation of blood low-density lipoproteins whereas high-density lipoprotein levels matched those of age-matched, chow-fed controls. Alcohol-induced constriction was only blunted by high-CLR dietary intake when high-CLR chow was taken for up to 8-12 and 18-23 weeks. However, alcohol-constriciton was not blunted when high-CLR chow intake lasted for longer times, such as 28-32 and 38-41 weeks. Thus, alcohol-induced constriction of rat middle cerebral arteries did not critically depend on the total blood CLR levels. Alcohol-induced constriction seemed unrelated to the natural, progressive elevation of the total blood CLR level in control- or high-CLR-fed animals over time. Thus, neither the exogenously nor endogenously driven increases in blood CLR could predict cerebral artery susceptibility to alcohol-induced constriction. However, we identified a temporal requirement for the protective effect of dietary CLR against alcohol, that could be governed by the young age of the high- CLR chow recipients (3 weeks of age) and/or the short duration of high-CLR chow feeding lasting for up to 23 weeks.

Influenza B viruses from different genetic backgrounds are variably impaired by neuraminidase inhibitor resistance-associated substitutions.

Identifying evolutionary routes to antiviral resistance among influenza viruses informs molecular-based resistance surveillance and clinical decisions. To improve antiviral management and understand whether clinically identified neuraminidase (NA) inhibitor (NAI) resistance-associated markers affect influenza B viruses of the Victoria- or Yamagata-lineages differentially, we generated a panel of NAI-resistant viruses (carrying E105K, G145E, R150K, D197N, I221 L/N/T/V, H273Y, N294S, or G407S substitutions; B numbering) in B/Brisbane/60/2008 (BR/08) and B/Phuket/3073/2013 (PH/13). In both backgrounds, I221 L/N/T/V resulted in reduced or highly reduced inhibition (HRI) by one to three currently available NAIs. D197N reduced inhibition by all NAIs in BR/08 but only by oseltamivir and peramivir in PH/13; R150K caused HRI by all NAIs in PH/13. Although PH/13 generally retained or enhanced NA activity in the presence of the substitutions, enzymatic activity in BR/08 was detrimentally affected. Similarly, substrate affinity and catalysis were relatively stable in PH/13, but not in the BR/08 variants. E105K, R150K, and D197N attenuated replication efficiency of BR/08 in vitro and in mice; only E105K had this effect in PH/13. Notably, the I221 L/N/T/V substitutions did not severely impair replication, particularly in PH/13. Overall, our data show differential effects of NA substitutions in representative Victoria- and Yamagata-lineage viruses, suggesting distinct evolution of these viruses caused variable fitness and NAI susceptibility profiles when similar key NA substitutions arise. Because the viruses harboring the I221 NA substitutions displayed undiminished fitness and are commonly reported, this position is likely to be the most clinically relevant marker for NAI resistance among contemporary influenza B viruses.

Gestational Age-Dependent Interplay between Endocannabinoid Receptors and Alcohol in Fetal Cerebral Arteries.

Alcohol (ethanol) is one of the most widely consumed drugs. Alcohol consumption by pregnant women may result in a range of fetal abnormalities termed fetal alcohol spectrum disorders (FASDs). The cerebrovascular system is emerging as a critical target of alcohol in the developing brain. We recently showed that three episodes of prenatal alcohol exposure resulting in 80 mg/dL alcohol in maternal blood during mid-pregnancy up-regulated anandamide-induced dilation of fetal cerebral arteries. Moreover, ethanol dilated fetal cerebral arteries via cannabinoid (CB) receptors. Whether a critical role of fetal cerebral artery CB system in responses to alcohol was maintained throughout the gestation, remains unknow. MAIN METHODS: Pregnant baboons (second trimester equivalent) were subjected to three episodes of either alcohol or control drink infusion via gavage. Cerebral arteries from mothers and near-term female fetuses were in vitro pressurized for diameter monitoring. KEY FINDINGS: Near-term fetal and maternal arteries exhibited similar ability to develop myogenic tone, to constrict in presence of 60 mM KCl, and to respond to 10 µM anandamide. Fetal and maternal arteries largely failed to dilate in presence of 63 mM ethanol. No differences were detected between arteries from control and alcohol-exposed baboon donors. Therefore, previously observed ethanol-induced dilation of fetal cerebral arteries and up-regulation of CB components in response to fetal alcohol exposure during mid-pregnancy was transient and disappeared by near-term.

Proteomic Analysis of Baboon Cerebral Artery Reveals Potential Pathways of Damage by Prenatal Alcohol Exposure.

Alcohol is one of the most widely misused substances in the world. Alcohol consumption by pregnant women often results in an array of fetal developmental abnormalities, but the damage to the fetus by alcohol remains poorly understood. The limited knowledge regarding the molecular targets of alcohol in the developing fetus constitutes one of the major obstacles in developing effective pharmacological interventions that could prevent fetal damage after alcohol consumption by pregnant women. The fetal cerebral artery is emerging as an important mediator of fetal cerebral damage by maternal alcohol drinking. In the present work, we conduct proteomics analysis of cerebral (basilar) artery lysates of near-term fetal baboons to search for protein targets of fetal alcohol exposure. Our study demonstrates that 3 episodes of binge alcohol exposure during the second trimester-equivalent of human pregnancy are sufficient to render profound changes in fetal cerebral artery proteome. These changes persisted, as they were detected in near-term fetuses. In particular, the relative abundance of 238 proteins differed significantly between control and alcohol-exposed fetuses. Enrichment analysis pointed at the group of metabolic activity proteins as a major class targeted by alcohol. Western blotting confirmed upregulation of the aldehyde dehydrogenase 6 family member A1 (ALDH6A1) in cerebral artery lysates from alcohol-exposed fetuses. This upregulation translated to greater ALDH activity of cerebral artery lysate of near-term fetuses following prenatal alcohol exposure when compared with controls.

Large conductance voltage- and calcium-gated potassium channels (BK) in cerebral artery myocytes of perinatal fetal primates share several major characteristics with the adult phenotype.

Large conductance voltage- and calcium-gated channels (BK) control fundamental processes, including smooth muscle contractility and artery diameter. We used a baboon (Papio spp) model of pregnancy that is similar to that of humans to characterize BK channels in the middle cerebral artery and its branches in near-term (165 dGa) primate fetuses and corresponding pregnant mothers. In cell-attached patches (K+pipette = 135 mM) on freshly isolated fetal cerebral artery myocytes, BK currents were identified by large conductance, and voltage- and paxilline-sensitive effects. Their calcium sensitivity was confirmed by a lower Vhalf (transmembrane voltage needed to reach half-maximal current) in inside-out patches at 30 versus 3 µM [Ca2+]free. Immunostaining against the BK channel-forming alpha subunit revealed qualitatively similar levels of BK alpha protein-corresponding fluorescence in fetal and maternal myocytes. Fetal and maternal BK currents recorded at 3 µM [Ca2+]free from excised membrane patches had similar unitary current amplitude, and Vhalf. However, subtle differences between fetal and maternal BK channel phenotypes were detected in macroscopic current activation kinetics. To assess BK function at the organ level, fetal and maternal artery branches were pressurized in vitro at 30 mmHg and probed with the selective BK channel blocker paxilline (1 µM). The degree of paxilline-induced constriction was similar in fetal and maternal arteries, yet the constriction of maternal arteries was achieved sooner. In conclusion, we present a first identification and characterization of fetal cerebral artery BK channels in myocytes from primates. Although differences in BK channels between fetal and maternal arteries exist, the similarities reported herein advance the idea that vascular myocyte BK channels are functional near-term, and thus may serve as pharmacological targets during the perinatal-neonatal period.

Tyrosine 450 in the Voltage- and Calcium-Gated Potassium Channel of Large Conductance Channel Pore-Forming (slo1) Subunit Mediates Cholesterol Protection against Alcohol-Induced Constriction of Cerebral Arteries.

Alcohol (ethanol) at physiologically relevant concentrations (<100 mM) constricts cerebral arteries via inhibition of voltage- and calcium-gated potassium channels of large conductance (BK) located in vascular smooth muscle (VSM). These channels consist of channel-forming slo1 (cbv1, KCNMA1) and accessory beta1 (KCNMB1) subunits. An increase in VSM cholesterol (CLR) via either dietary CLR intake or in vitro CLR enrichment was shown to protect against endothelium-independent, alcohol-induced constriction of cerebral arteries. The molecular mechanism(s) of this protection remains unknown. Here, we demonstrate that CLR enrichment of de-endothelialized middle cerebral arteries (MCAs) of rat increased CLR content in the VSM in a concentration-dependent manner. CLR enrichment blunted MCA constriction evoked by 18-75 mM but not by 100 mM alcohol. MCA enrichment with coprostanol (COPR) also blunted vasoconstriction by 50 mM alcohol, despite the fact that COPR and CLR differ in their ability to modify several major physical properties of the bilayer. CLR protection against 50 but not 100 mM alcohol was also observed in C57BL/6 and KCNMB1 knockout (KO) mice. Permeabilization of KCNMA1 KO MCAs with Y450Fcbv1 totally ablated CLR, but not COPR protection against vasoconstriction by 50 mM alcohol. Thus, CLR and alcohol interact at the level of the BK channel slo1 subunit, with Y450 being critical for CLR protection against alcohol-induced vasoconstriction. We document for the first time a functional competition between CLR and alcohol in regulating cerebral artery diameter and a critical role of a single amino acid within the BK channel pore-forming subunit in controlling CLR-alcohol interaction at the organ level.

Statin therapy exacerbates alcohol-induced constriction of cerebral arteries via modulation of ethanol-induced BK channel inhibition in vascular smooth muscle.

Statins constitute the most commonly prescribed drugs to decrease cholesterol (CLR). CLR is an important modulator of alcohol-induced cerebral artery constriction (AICAC). Using rats on a high CLR diet (2% CLR) we set to determine whether atorvastatin administration (10mg/kg daily for 18-23weeks) modified AICAC. Middle cerebral arteries were pressurized in vitro at 60mmHg and AICAC was evoked by 50mM ethanol, that is within the range of blood alcohol detected in humans following moderate-to-heavy drinking. AICAC was evident in high CLR+atorvastatin group but not in high CLR diet+placebo. Statin exacerbation of AICAC persisted in de-endothelialized arteries, and was blunted by CLR enrichment in vitro. Fluorescence imaging of filipin-stained arteries showed that atorvastatin decreased vascular smooth muscle (VSM) CLR when compared to placebo, this difference being reduced by CLR enrichment in vitro. Voltage- and calcium-gated potassium channels of large conductance (BK) are known VSM targets of ethanol, with their beta1 subunit being necessary for ethanol-induced channel inhibition and resulting AICAC. Ethanol-induced BK inhibition in excised membrane patches from freshly isolated myocytes was exacerbated in the high CLR diet+atorvastatin group when compared to high CLR diet+placebo. Unexpectedly, atorvastatin decreased the amount and function of BK beta1 subunit as documented by immunofluorescence imaging and functional patch-clamp studies. Atorvastatin exacerbation of ethanol-induced BK inhibition disappeared upon artery CLR enrichment in vitro. Our study demonstrates for the first time statin's ability to exacerbate the vascular effect of a widely consumed drug of abuse, this exacerbation being driven by statin modulation of ethanol-induced BK channel inhibition in the VSM via CLR-mediated mechanism.

Differential distribution and functional impact of BK channel beta1 subunits across mesenteric, coronary, and different cerebral arteries of the rat.

Large conductance, Ca2+i- and voltage-gated K+ (BK) channels regulate myogenic tone and, thus, arterial diameter. In smooth muscle (SM), BK channels include channel-forming α and auxiliary ß1 subunits. BK ß1 increases the channel's Ca2+ sensitivity, allowing BK channels to negatively feedback on depolarization-induced Ca2+ entry, oppose SM contraction and favor vasodilation. Thus, endothelial-independent vasodilation can be evoked though targeting of SM BK ß1 by endogenous ligands, including lithocholate (LCA). Here, we investigated the expression of BK ß1 across arteries of the cerebral and peripheral circulations, and the contribution of such expression to channel function and BK ß1-mediated vasodilation. Data demonstrate that endothelium-independent, BK ß1-mediated vasodilation by LCA is larger in coronary (CA) and basilar (BA) arteries than in anterior cerebral (ACA), middle cerebral (MCA), posterior cerebral (PCA), and mesenteric (MA) arteries, all arterial segments having a similar diameter. Thus, differential dilation occurs in extracranial arteries which are subjected to similar vascular pressure (CA vs. MA) and in arteries that irrigate different brain regions (BA vs. ACA, MCA, and PCA). SM BK channels from BA and CA displayed increased basal activity and LCA responses, indicating increased BK ß1 functional presence. Indeed, in the absence of detectable changes in BK α, BA and CA myocytes showed an increased location of BK ß1 in the plasmalemma/subplasmalemma. Moreover, these myocytes distinctly showed increased BK ß1 messenger RNA (mRNA) levels. Supporting a major role of enhanced BK ß1 transcripts in artery dilation, LCA-induced dilation of MCA transfected with BK ß1 complementary DNA (cDNA) was as high as LCA-induced dilation of untransfected BA or CA.

Distinct mechanisms underlying cholesterol protection against alcohol-induced BK channel inhibition and resulting vasoconstriction.

Alcohol (ethanol) at concentrations reached in blood following moderate to heavy drinking (30-80mM) reduces cerebral artery diameter via inhibition of voltage- and calcium-gated potassium channels of large conductance (BK) in cerebral artery smooth muscle. These channels consist of channel-forming α and regulatory ß1 subunits. A high-cholesterol diet protects against ethanol-induced constriction via accumulation of cholesterol within the vasculature. The molecular mechanisms of this protection remain unknown. In the present work, we demonstrate that in vitro cholesterol enrichment of rat middle cerebral arteries significantly increased cholesterol within arterial tissues and blunted constriction by 50mM of ethanol. Ethanol-induced BK channel inhibition in inside-out patches excised from freshly isolated cerebral artery myocytes was also abolished by cholesterol enrichment. Enrichment of arteries with enantiomeric cholesterol (ent-cholesterol) also blunted BK channel inhibition and cerebral artery constriction in response to ethanol. The similar protection of cholesterol and ent-cholesterol against ethanol action indicates that this protection does not require protein site(s) that specifically sense natural cholesterol. Cholesterol-driven protection against ethanol-induced BK channel inhibition and vasoconstriction was replicated in myocytes and middle cerebral arteries of C57BL/6 mice. BK ß1 subunits are known to regulate vascular diameter and its modification by ethanol. However, blunting of an ethanol effect by in vitro cholesterol enrichment was observed in arteries and myocyte membrane patches from BK ß1 (KCNMB1) knockout mice. Thus, BK ß1 subunits are not needed for cholesterol protection against ethanol effect on BK channel function and cerebral artery diameter.

Age-Dependent Susceptibility to Alcohol-Induced Cerebral Artery Constriction.

BACKGROUND: Age has been recognized as an important contributor into susceptibility to alcohol-driven pathology. PURPOSE: We aimed at determining whether alcohol-induced constriction of cerebral arteries was age-dependent. STUDY DESIGN: We used rat middle cerebral artery (MCA) in vitro diameter monitoring, patch-clamping and fluorescence labeling of myocytes to study an age-dependent increase in the susceptibility to alcohol in 3 (50 g), 8 (250 g), and 15 (440 g) weeks-old rats. RESULTS: An age-dependent increase in alcohol-induced constriction of MCA could be observed in absence of endothelium, which is paralleled by an age-dependent increase in both protein level of the calcium-/voltage-gated potassium channel of large conductance (BK) accessory ß1 subunit and basal BK channel activity. Ethanol-induced BK channel inhibition is increased with age. CONCLUSIONS: We demonstrate an increased susceptibility of MCA to ethanol-induced constriction in a period equivalent to adolescence and early adulthood when compared to pre-adolescence. Our work suggests that BK ß1 constitutes a significant contributor to age-dependent changes in the susceptibility of cerebral arteries to ethanol.