Impairment of endothelial glycocalyx in atherosclerosis and obesity.

Endothelial glycocalyx is a negatively charged gel-like layer located on the apical surface of endothelial cells. It serves as a selective two-way physical barrier between the flowing blood and the endothelium, which regulates the access of macromolecules and of blood cells to the endothelial surface. In addition, endothelial glycocalyx plays a major role in sensing mechanical signals generated by the blood flow and transducing these signals to maintain endothelial functions; Thus, dysfunction or disruption of endothelial glycocalyx in pathological condition leads to endothelial dysfunction and contributes to the development of vascular diseases. In this review, we discuss the impact of atherosclerosis with the following viewpoints: (i) hypercholesterolemic effects on endothelial glycocalyx degradation in animal models and human patients, (ii) disruption of endothelial glycocalyx by atherogenic lipoproteins, (iii) proatherogenic disturbed flow effects on endothelial glycocalyx degradation, (iv) pathological consequences of the loss of glycocalyx integrity in atherogenesis, and (v) therapeutic effect of glycocalyx supplementation on atherosclerosis development. Additionally, we also discuss recent studies in pathological effects of obesity on the disruption of endothelial glycocalyx.

Cholesterol-Induced Suppression of Endothelial Kir Channels Is a Driver of Impairment of Arteriolar Flow-Induced Vasodilation in Humans.

Dyslipidemia-induced endothelial dysfunction is an important factor in the progression of cardiovascular disease; however, the underlying mechanisms are unclear. Our recent studies demonstrated that flow-induced vasodilation (FIV) is regulated by inwardly rectifying K+ channels (Kir2.1) in resistance arteries. Furthermore, we showed that hypercholesterolemia inhibits Kir2.1-dependent vasodilation. In this study, we introduced 2 new mouse models: (1) endothelial-specific deletion of Kir2.1 to demonstrate the role of endothelial Kir2.1 in FIV and (2) cholesterol-insensitive Kir2.1 mutant to determine the Kir2.1 regulation in FIV under hypercholesterolemia. FIV was significantly reduced in endothelial-specific Kir2.1 knock-out mouse mesenteric arteries compared with control groups. In cholesterol-insensitive Kir2.1 mutant mice, Kir2.1 currents were not affected by cyclodextrin and FIV was restored in cells and arteries, respectively, with a hypercholesterolemic background. To extend our observations to humans, 16 healthy subjects were recruited with LDL (low-density lipoprotein)-cholesterol ranging from 51 to 153 mg/dL and FIV was assessed in resistance arteries isolated from gluteal adipose. Resistance arteries from participants with >100 mg/dL LDL (high-LDL) exhibited reduced FIV as compared with those participants with <100 mg/dL LDL (low-LDL). A significant negative correlation was observed between LDL cholesterol and FIV in high-LDL. Expressing dominant-negative Kir2.1 in endothelium blunted FIV in arteries from low-LDL but had no further effect on FIV in arteries from high-LDL. The Kir2.1-dependent vasodilation more negatively correlated to LDL cholesterol in high-LDL. Overexpressing wild-type Kir2.1 in endothelium fully recovered FIV in arteries from participants with high-LDL. Our data suggest that cholesterol-induced suppression of Kir2.1 is a major mechanism underlying endothelial dysfunction in hypercholesterolemia.

Cholesterol-induced suppression of Kir2 channels is mediated by decoupling at the inter-subunit interfaces.

Cholesterol is a major regulator of multiple types of ion channels. Although there is increasing information about cholesterol binding sites, the molecular mechanisms through which cholesterol binding alters channel function are virtually unknown. In this study, we used a combination of Martini coarse-grained simulations, a network theory-based analysis, and electrophysiology to determine the effect of cholesterol on the dynamic structure of the Kir2.2 channel. We found that increasing membrane cholesterol reduced the likelihood of contact between specific regions of the cytoplasmic and transmembrane domains of the channel, most prominently at the subunit-subunit interfaces of the cytosolic domains. This decrease in contact was mediated by pairwise interactions of specific residues and correlated to the stoichiometry of cholesterol binding events. The predictions of the model were tested by site-directed mutagenesis of two identified residues-V265 and H222-and high throughput electrophysiology.

The Membrane Cholesterol Modulates the Interaction Between 17-ßEstradiol and the BK Channel.

BK channels are composed by the pore forming α subunit and, in some tissues, is associated with different accessory ß subunits. These proteins modify the biophysical properties of the channel, amplifying the range of BK channel activation according to the physiological context. In the vascular cells, the pore forming BKα subunit is expressed with the ß1 subunit, where they play an essential role in the modulation of arterial tone and blood pressure. In eukaryotes, cholesterol is a structural lipid of the cellular membrane. Changes in the ratio of cholesterol content in the plasma membrane (PM) regulates the BK channel activation altering its open probability, and hence, vascular contraction. It has been shown that the estrogen 17ß-Estradiol (E2) causes a vasodilator effect in vascular cells, inducing a leftward shift in the V0.5 of the GV curve. Here, we evaluate whether changes in the membrane cholesterol concentration modify the effect that E2 induces on the BKα/ß1 channel activity. Using binding and electrophysiology assays after cholesterol depletion or enrichment, we show that the cholesterol enrichment significantly decreases the expression of the α subunit, while cholesterol depletion increased the expression of that α subunit. Additionally, we demonstrated that changes in the membrane cholesterol cause the loss of the modulatory effect of E2 on the BKα/ß1 channel activity, without affecting the E2 binding to the complex. Our data suggest that changes in membrane cholesterol content could affect channel properties related to the E2 effect on BKα/ß1 channel activity. Finally, the results suggest that an optimal membrane cholesterol content is essential for the activation of BK channels through the ß1 subunit.

Activation of BK Channel Contributes to PL-Induced Mesenchymal Stem Cell Migration.

Due to their capacity to proliferate, migrate, and differentiate, mesenchymal stem cells (MSCs) are considered to be good candidates for regenerative medicine applications. The mechanisms underlying proliferation and differentiation of MSCs have been studied. However, much less is known about the mechanisms regulating the migration of MSCs. Platelet lysate (PL), a supplement used to promote cell expansion, has been shown to promote MSCs migration; however, the underlying mechanism are unknown. Here, by using adipose-derived rat MSCs (rMSCs) and the scratch assay in the absence and presence of various BK channels modulators, we evaluated the role of BK channels in mediating the PL-stimulated migration of rMSCs. We found that 5% PL increased rMSCs migration, and this effect was blocked by the addition of the BK channel selective antagonist Iberiotoxin (IBTX). In the absence of PL, the BK channel agonist NS1619, stimulated rMSCs migration to similar level as 5% PL. Addition of both NS1619 and 5% PL resulted in an increase in rMSCs migration, that was higher than when either one was added individually. From whole-cell recordings, it was found that the addition of 5% PL increased the magnitude of BK current density. By using Western blot and flow cytometry, it was found that PL did not affect the expression of BK channels. Together, our results indicate that as shown in other cell types, activation of BK channels by themselves also promote rMSC migration, and show that activation of BK channels contribute to the observed PL-induced increase in migration of rMSC.

The molecular nature of the 17ß-Estradiol binding site in the voltage- and Ca2+-activated K+ (BK) channel ß1 subunit.

The accessory ß1 subunit modulates the Ca2+- and voltage-activated K+ (BK) channel gating properties mainly by increasing its apparent Ca2+ sensitivity. ß1 plays an important role in the modulation of arterial tone and blood pressure by vascular smooth muscle cells (SMCs). 17ß-estradiol (E2) increases the BK channel open probability (Po) in SMCs, through a ß1 subunit-dependent modulatory effect. Here, using molecular modeling, bioinformatics, mutagenesis, and electrophysiology, we identify a cluster of hydrophobic residues in the second transmembrane domain of the ß1 subunit, including the residues W163 and F166, as the binding site for E2. We further show that the increase in Po induced by E2 is associated with a stabilization of the voltage sensor in its active configuration and an increase in the coupling between the voltage sensor activation and pore opening. Since ß1 is a key molecular player in vasoregulation, the findings reported here are of importance in the design of novel drugs able to modulate BK channels.

Characteristics and long-term outcomes of advanced pleural mesothelioma in Latin America (MeSO-CLICaP).

BACKGROUND: Malignant pleural mesothelioma (MPM) is an aggressive tumor, associated with poor prognosis. There is a lack of information about the clinical and pathological features related with survival in the Latin American population. METHODS: The MeSO-CLICaP registry identified 302 patients with advanced MPM diagnosed and treated between January 2008 and March 2016. The Cox model was applied to determine the variables associated with survival. A random forest tree model was built to predict the response to first-line chemotherapy among Latin American patients. RESULTS: The median age was 61.1 years (SD 10.6 years), 191 (63.2%) were men, 65.9% were ever smokers, and 38.7% had previous exposure to asbestos. A total of 237 (78.5%) had epithelioid tumors, and 188 (62.3%) and 114 (37.7%) cases had stage III or IV MPM, respectively. A total of 49 patients (16.2%) underwent pleurectomy, 57 (18.9%) received radiotherapy, and 279 patients received first-line platinum-based chemotherapy. The overall response rate to first-line chemotherapy was 40.4%, progression-free survival to first-line treatment was 5.7 months (95% CI 4.9-6.5), and 63 (20.8%) patients had pemetrexed maintenance. The median overall survival was 16.8 months (95% CI 13.0-20.5), and multivariate analysis found that stage (P = 0.013), and pleurodesis (P = 0.048), were independent prognostic factors for first-line overall survival. The model to predict response to first-line chemotherapy obtained a 0.98 area under the curve, a sensitivity of 93%, and a specificity of 95% for detecting responders and non-responders. CONCLUSION: This study identifies factors associated with clinical benefit from chemotherapy among advanced MPM Latin American patients, emphasizing the impact of histology and the clinical benefit of chemotherapy on outcomes.

EGFR exon 20 insertion in lung adenocarcinomas among Hispanics (geno1.2-CLICaP).

OBJECTIVES: Contrasting other EGFR mutations (EGFRm) in lung adenocarcinomas, insertions in exon 20 (exon20ins) are generally associated with resistance to targeted therapy, limiting therapeutic options and impoverishing the prognosis compared to other EGFRm. We sought to extensively characterize exon20ins from a large cohort of lung adenocarcinomas in Hispanic patients. MATERIALS AND METHODS: This was a region-wide, observational longitudinal cohort study to evaluate characteristics and outcomes of patients with exon20ins in lung adenocarcinoma, based on a secondary analysis of electronic records from the Geno1.2-CLICaP Platform and extended genotype testing. Patients from six Latin-American countries were included (Argentina, Colombia, Costa Rica, Ecuador, Panama, and Mexico). Data obtained included the molecular spectrum (extended genotyping for mutations in BRAF, NRAS, PIK3CA, Her2 and MEK1, as well as for EGFR amplification, ALK and PD-L1 protein expression), clinic-pathologic characteristics, prevalence and outcomes to therapeutic approach. RESULTS AND CONCLUSIONS: 4.005 patients diagnosed with stage III/IV lung adenocarcinoma from 2011 to 2016 were initially screened. Among these, 88 patients had a confirmed exon20 in. and were included; median age was 66-years, 62.5% were females, 64% were never smokers and 39% presented with brain metastases. The H773insH variant was the most frequent, making up 21.6% of cases. A common EGFRm was concomitantly found in 36.4% (del19/L858R), and 8% (G719X/L861Q/S768I) of cases. Five cases had additional mutations in PI3K, KRAS and MEK1, 26% had EGFR amplification and 81.7% had PD-L1 expression 1-50%. Overall response rate to first-line therapy was 28% and overall survival was 16.4 months. Prognosis was positively influenced by the concomitant presence of common EGFRm and response to first-line. Our results suggest that patients with EGFR exon20ins have similar clinical characteristics to those with common EGFRm but a poorer prognosis. Last, the mean PD-L1 expression in this population seems higher than for patients with common EGFRm.

Pharmacological consequences of the coexpression of BK channel α and auxiliary ß subunits.

Coded by a single gene (Slo1, KCM) and activated by depolarizing potentials and by a rise in intracellular Ca(2+) concentration, the large conductance voltage- and Ca(2+)-activated K(+) channel (BK) is unique among the superfamily of K(+) channels. BK channels are tetramers characterized by a pore-forming α subunit containing seven transmembrane segments (instead of the six found in voltage-dependent K(+) channels) and a large C terminus composed of two regulators of K(+) conductance domains (RCK domains), where the Ca(2+)-binding sites reside. BK channels can be associated with accessory ß subunits and, although different BK modulatory mechanisms have been described, greater interest has recently been placed on the role that the ß subunits may play in the modulation of BK channel gating due to its physiological importance. Four ß subunits have currently been identified (i.e., ß1, ß2, ß3, and ß4) and despite the fact that they all share the same topology, it has been shown that every ß subunit has a specific tissue distribution and that they modify channel kinetics as well as their pharmacological properties and the apparent Ca(2+) sensitivity of the α subunit in different ways. Additionally, different studies have shown that natural, endogenous, and synthetic compounds can modulate BK channels through ß subunits. Considering the importance of these channels in different pathological conditions, such as hypertension and neurological disorders, this review focuses on the mechanisms by which these compounds modulate the biophysical properties of BK channels through the regulation of ß subunits, as well as their potential therapeutic uses for diseases such as those mentioned above.