Erythrocyte-Derived microRNAs: Emerging Players in Cardiovascular and Metabolic Disease.

Recent studies have demonstrated a novel function of red blood cells (RBCs) beyond their classical role as gas transporters, that is, RBCs undergo functional alterations in cardiovascular and metabolic disease, and RBC dysfunction is associated with hypertension and the development of cardiovascular injury in type 2 diabetes, heart failure, preeclampsia, familial hypercholesterolemia/dyslipidemia, and COVID-19. The underlying mechanisms include decreased nitric oxide bioavailability, increased arginase activity, and reactive oxygen species formation. Of interest, RBCs contain diverse and abundant micro (mi)RNAs. miRNA expression pattern in RBCs reflects the expression in the whole blood, serum, and plasma. miRNA levels in RBCs have been found to be altered in various cardiovascular and metabolic diseases, which contributes to the development of cardiovascular complications. Evidence has shown that RBC-derived miRNAs interact with the cardiovascular system via extracellular vesicles and argonaute RISC catalytic component 2 as carriers. Alteration of RBC-to-vascular communication via miRNAs may serve as potential disease mechanism for vascular complications. The present review summarizes RBCs and their released miRNAs as potential mediators of cardiovascular injury. We further focus on the possible mechanisms by which RBC-derived miRNAs regulate cardiovascular function. A better understanding of the function of RBC-derived miRNAs will increase insights into the disease mechanism and potential targets for the treatment of cardiovascular complications.

Extracellular vesicles and their non-coding RNA cargos: Emerging players in cardiovascular disease.

Extracellular vesicles (EVs), including exosomes, microvesicles and apoptotic bodies, have recently received attention as essential mechanisms for cell-to-cell communication in cardiovascular disease. EVs can be released from different types of cells, including endothelial cells, smooth muscle cells, cardiac cells, fibroblasts, platelets, adipocytes, immune cells and stem cells. Non-coding (nc)RNAs as EV cargos have recently been investigated in the cardiovascular system. Up- or downregulated ncRNAs in EVs have been shown to play a crucial role in various cardiovascular diseases. Communication via EV-derived ncRNAs can occur between cells of the same type and between different types of cells involved in the pathophysiology of cardiovascular disease. In the present review, we highlight the important aspects of diverse cell-derived EVs and their ncRNA cargos as disease mediators and potential therapeutic targets in atherosclerosis, coronary artery disease, ischaemic heart disease and cardiac fibrosis. In addition, we summarize the potential of EV-derived ncRNAs in the treatment of cardiovascular disease. Finally, we discuss the different methods for EV isolation and characterization. A better understanding of the specific role of EVs and their ncRNA cargos in the regulation of cardiovascular (dys)function will be of importance for the development of diagnostic and therapeutic tools for cardiovascular disease.

The red blood cell as a mediator of endothelial dysfunction in patients with familial hypercholesterolemia and dyslipidemia.

BACKGROUND: Patients with familial hypercholesterolemia (FH) display high levels of low-density lipoprotein cholesterol (LDL-c), endothelial dysfunction, and increased risk of premature atherosclerosis. We have previously shown that red blood cells (RBCs) from patients with type 2 diabetes induce endothelial dysfunction through increased arginase 1 and reactive oxygen species (ROS). OBJECTIVE: To test the hypothesis that RBCs from patients with FH (FH-RBCs) and elevated LDL-c induce endothelial dysfunction. METHODS AND RESULTS: FH-RBCs and LDL-c >5.0 mM induced endothelial dysfunction following 18-h incubation with isolated aortic rings from healthy rats compared to FH-RBCs and LDL-c <2.5 mM or RBCs from healthy subjects (H-RBCs). Inhibition of vascular but not RBC arginase attenuated the degree of endothelial dysfunction induced by FH-RBCs and LDL-c >5.0 mM. Furthermore, arginase 1 but not arginase 2 was elevated in the vasculature of aortic segments after incubation with FH-RBCs and LDL-c >5.0 mM. A superoxide scavenger, present throughout the 18-h incubation, attenuated the degree of endothelial dysfunction induced by FH-RBCs and LDL-c >5.0 mM. ROS production was elevated in these RBCs in comparison with H-RBCs. Scavenging of vascular ROS through various antioxidants also attenuated the degree of endothelial dysfunction induced by FH-RBCs and LDL-c >5.0 mM. This was corroborated by an increase in the lipid peroxidation product 4-hydroxynonenal. Lipidomic analysis of RBC lysates did not reveal any significant changes across the groups. CONCLUSION: FH-RBCs induce endothelial dysfunction dependent on LDL-c levels via arginase 1 and ROS-dependent mechanisms.

Anti-inflammatory effects and improved metabolic derangements in ob/ob mice by a newly synthesized prenylated benzopyran with pan-PPAR activity.

BACKGROUND AND PURPOSE: Selective peroxisome proliferator-activated receptors (PPARs) are widely used to treat metabolic complications; however, the limited effect of PPARα agonists on glucose metabolism and the adverse effects associated with selective PPARγ activators have stimulated the development of novel pan-PPAR agonists to treat metabolic disorders. Here, we synthesized a new prenylated benzopyran (BP-2) and evaluated its PPAR-activating properties, anti-inflammatory effects and impact on metabolic derangements. EXPERIMENTAL APPROACH: BP-2 was used in transactivation assays to evaluate its agonism to PPARα, PPARß/δ and PPARγ. A parallel-plate flow chamber was employed to investigate its effect on TNFα-induced leukocyte-endothelium interactions. Flow cytometry and immunofluorescence were used to determine its effects on the expression of endothelial cell adhesion molecules (CAMs) and chemokines and p38-MAPK/NF-κB activation. PPARs/RXRα interactions were determined using a gene silencing approach. Analysis of its impact on metabolic abnormalities and inflammation was performed in ob/ob mice. KEY RESULTS: BP-2 displayed strong PPARα activity, with moderate and weak activity against PPARß/δ and PPARγ, respectively. In vitro, BP-2 reduced TNFα-induced endothelial ICAM-1, VCAM-1 and fractalkine/CX3CL1 expression, suppressed mononuclear cell arrest via PPARß/δ-RXRα interactions and decreased p38-MAPK/NF-κB activation. In vivo, BP-2 improved the circulating levels of glucose and triglycerides in ob/ob mice, suppressed T-lymphocyte/macrophage infiltration and proinflammatory markers in the liver and white adipose tissue, but increased the expression of the M2-like macrophage marker CD206. CONCLUSION AND IMPLICATIONS: BP-2 emerges as a novel pan-PPAR lead candidate to normalize glycemia/triglyceridemia and minimize inflammation in metabolic disorders, likely preventing the development of further cardiovascular complications.

Erythrocytes from patients with ST-elevation myocardial infarction induce cardioprotection through the purinergic P2Y13 receptor and nitric oxide signaling.

Red blood cells (RBCs) are suggested to play a role in cardiovascular regulation by exporting nitric oxide (NO) bioactivity and ATP under hypoxia. It remains unknown whether such beneficial effects of RBCs are protective in patients with acute myocardial infarction. We investigated whether RBCs from patients with ST-elevation myocardial infarction (STEMI) protect against myocardial ischemia-reperfusion injury and whether such effect involves NO and purinergic signaling in the RBCs. RBCs from patients with STEMI undergoing primary coronary intervention and healthy controls were administered to isolated rat hearts subjected to global ischemia and reperfusion. Compared to RBCs from healthy controls, RBCs from STEMI patients reduced myocardial infarct size (30 ± 12% RBC healthy vs. 11 ± 5% RBC STEMI patients, P < 0.001), improved recovery of left-ventricular developed pressure and dP/dt and reduced left-ventricular end-diastolic pressure in hearts subjected to ischemia-reperfusion. Inhibition of RBC NO synthase with L-NAME or soluble guanylyl cyclase (sGC) with ODQ, and inhibition of cardiac protein kinase G (PKG) abolished the cardioprotective effect. Furthermore, the non-selective purinergic P2 receptor antagonist PPADS but not the P1 receptor antagonist 8PT attenuated the cardioprotection induced by RBCs from STEMI patients. The P2Y13 receptor was expressed in RBCs and the cardioprotection was abolished by the P2Y13 receptor antagonist MRS2211. By contrast, perfusion with PPADS, L-NAME, or ODQ prior to RBCs administration failed to block the cardioprotection induced by RBCs from STEMI patients. Administration of RBCs from healthy subjects following pre-incubation with an ATP analog reduced infarct size from 20 ± 6 to 7 ± 2% (P < 0.001), and this effect was abolished by ODQ and MRS2211. This study demonstrates a novel function of RBCs in STEMI patients providing protection against myocardial ischemia-reperfusion injury through the P2Y13 receptor and the NO-sGC-PKG pathway.

SGLT-2 (Sodium-Glucose Cotransporter 2) Inhibition Reduces Ang II (Angiotensin II)-Induced Dissecting Abdominal Aortic Aneurysm in ApoE (Apolipoprotein E) Knockout Mice.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is a pathological condition of permanent vessel dilatation that predisposes to the potentially fatal consequence of aortic rupture. SGLT-2 (sodium-glucose cotransporter 2) inhibitors have emerged as powerful pharmacological tools for type 2 diabetes mellitus treatment. Beyond their glucose-lowering effects, recent studies have shown that SGLT-2 inhibitors reduce cardiovascular events and have beneficial effects on several vascular diseases such as atherosclerosis; however, the potential effects of SGLT-2 inhibition on AAA remain unknown. This study evaluates the effect of oral chronic treatment with empagliflozin-an SGLT-2 inhibitor-on dissecting AAA induced by Ang II (angiotensin II) infusion in apoE (apolipoprotein E)-/- mice. Approach and Results: Empagliflozin treatment significantly reduced the Ang II-induced increase in maximal suprarenal aortic diameter in apoE-/- mice independently of blood pressure effects. Immunohistochemistry analysis revealed that empagliflozin diminished Ang II-induced elastin degradation, neovessel formation, and macrophage infiltration at the AAA lesion. Furthermore, Ang II infusion resulted in a marked increase in the expression of chemokines (CCL-2 [chemokine (C-C motif) ligand 2] and CCL-5 [chemokine (C-C motif) ligand 5]), VEGF (vascular endothelial growth factor), and MMP (matrix metalloproteinase)-2 and MMP-9 in suprarenal aortic walls of apoE-/- mice, and all were reduced by empagliflozin cotreatment. Western blot analysis revealed that p38 MAPK (p38 mitogen-activated protein kinase) and NF-κB (nuclear factor-κB) activation was also reduced in the suprarenal aortas of apoE-/- mice cotreated with empagliflozin. Finally, in vitro studies in human aortic endothelial cells and macrophages showed that empagliflozin inhibited leukocyte-endothelial cell interactions and release of proinflammatory chemokines. CONCLUSIONS: Pharmacological inhibition of SGLT-2 by empagliflozin inhibits AAA formation. SGLT-2 inhibition might represent a novel promising therapeutic strategy to prevent AAA progression.

Polycerasoidol, a Natural Prenylated Benzopyran with a Dual PPARα/PPARγ Agonist Activity and Anti-inflammatory Effect.

Dual peroxisome proliferator-activated receptor-α/γ (PPARα/γ) agonists regulate both lipid and glucose homeostasis under different metabolic conditions and can exert anti-inflammatory activity. We investigated the potential dual PPARα/γ agonism of prenylated benzopyrans polycerasoidol (1) and polycerasoidin (2) and their derivatives for novel drug development. Nine semisynthetic derivatives were prepared from the natural polycerasoidol (1) and polycerasoidin (2), which were evaluated for PPARα, -γ, -δ and retinoid X receptor-α activity in transactivation assays. Polycerasoidol (1) exhibited potent dual PPARα/γ agonism and low cytotoxicity. Structure-activity relationship studies revealed that a free phenol group at C-6 and a carboxylic acid at C-9' were key features for dual PPARα/γ agonism activity. Molecular modeling indicated the relevance of these groups for optimal ligand binding to the PPARα and PPARγ domains. In addition, polycerasoidol (1) exhibited a potent anti-inflammatory effect by inhibiting mononuclear leukocyte adhesion to the dysfunctional endothelium in a concentration-dependent manner via RXRα/PPARγ interactions. Therefore, polycerasoidol (1) can be considered a hit-to-lead molecule for the further development of novel dual PPARα/γ agonists capable of preventing cardiovascular events associated with metabolic disorders.

Vitamin D Receptor Activation Reduces Angiotensin-II-Induced Dissecting Abdominal Aortic Aneurysm in Apolipoprotein E-Knockout Mice.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is a vascular disorder characterized by chronic inflammation of the aortic wall. Low concentrations of vitamin D3 are associated with AAA development; however, the potential direct effect of vitamin D3 on AAA remains unknown. This study evaluates the effect of oral treatment with the vitamin D3 receptor (VDR) ligand, calcitriol, on dissecting AAA induced by angiotensin-II (Ang-II) infusion in apoE(-/-) mice. APPROACH AND RESULTS: Oral treatment with calcitriol reduced Ang-II-induced dissecting AAA formation in apoE(-/-) mice, which was unrelated to systolic blood pressure or plasma cholesterol concentrations. Immunohistochemistry and reverse-transcription polymerase chain reaction analysis demonstrated a significant increase in macrophage infiltration, neovessel formation, matrix metalloproteinase-2 and matrix metalloproteinase-9, chemokine (CCL2 [(C-C motif) ligand 2], CCL5 [(C-C motif) ligand 5], and CXCL1 [(C-X-C motif) ligand 1]) and vascular endothelial growth factor expression in suprarenal aortic walls of apoE(-/-) mice infused with Ang-II, and all were significantly reduced by cotreatment with calcitriol. Phosphorylation of extracellular signal-regulated kinases 1/2, p38 mitogen-activated protein kinase, and nuclear factor-κB was also decreased in the suprarenal aortas of apoE(-/-) mice cotreated with calcitriol. These effects were accompanied by a marked increase in VDR-retinoid X receptor (RXR) interaction in the aortas of calcitriol-treated mice. In vitro, VDR activation by calcitriol in human endothelial cells inhibited Ang-II-induced leukocyte-endothelial cell interactions, morphogenesis, and production of endothelial proinflammatory and angiogenic chemokines through VDR-RXR interactions, and knockdown of VDR or RXR abolished the inhibitory effects of calcitriol. CONCLUSIONS: VDR activation reduces dissecting AAA formation induced by Ang-II in apoE(-/-) mice and may constitute a novel therapeutic strategy to prevent AAA progression.

Stimulation of Erythrocyte Soluble Guanylyl Cyclase Induces cGMP Export and Cardioprotection in Type 2 Diabetes.

Reduced nitric oxide (NO) bioactivity in red blood cells (RBCs) is critical for augmented myocardial ischemia-reperfusion injury in type 2 diabetes. This study identified the nature of "NO bioactivity" by stimulating the intracellular NO receptor soluble guanylyl cyclase (sGC) in RBCs. sGC stimulation in RBCs from patients with type 2 diabetes increased export of cyclic guanosine monophosphate from RBCs and activated cardiac protein kinase G, thereby attenuating ischemia-reperfusion injury. These results provide novel insight into RBC signaling by identifying cyclic guanosine monophosphate from RBC as a mediator of protection against cardiac ischemia-reperfusion injury induced by sGC stimulation in RBCs.

Hypoxic erythrocytes mediate cardioprotection through activation of soluble guanylate cyclase and release of cyclic GMP.

Red blood cells (RBCs) mediate cardioprotection via nitric oxide-like bioactivity, but the signaling and the identity of any mediator released by the RBCs remains unknown. We investigated whether RBCs exposed to hypoxia release a cardioprotective mediator and explored the nature of this mediator. Perfusion of isolated hearts subjected to ischemia-reperfusion with extracellular supernatant from mouse RBCs exposed to hypoxia resulted in improved postischemic cardiac function and reduced infarct size. Hypoxia increased extracellular export of cyclic guanosine monophosphate (cGMP) from mouse RBCs, and exogenous cGMP mimicked the cardioprotection induced by the supernatant. The protection induced by hypoxic RBCs was dependent on RBC-soluble guanylate cyclase and cGMP transport and was sensitive to phosphodiesterase 5 and activated cardiomyocyte protein kinase G. Oral administration of nitrate to mice to increase nitric oxide bioactivity further enhanced the cardioprotective effect of hypoxic RBCs. In a placebo-controlled clinical trial, a clear cardioprotective, soluble guanylate cyclase-dependent effect was induced by RBCs collected from patients randomized to 5 weeks nitrate-rich diet. It is concluded that RBCs generate and export cGMP as a response to hypoxia, mediating cardioprotection via a paracrine effect. This effect can be further augmented by a simple dietary intervention, suggesting preventive and therapeutic opportunities in ischemic heart disease.

Erythrocytes Induce Vascular Dysfunction in COVID-19.

Current knowledge regarding mechanisms underlying cardiovascular complications in patients with COVID-19 is limited and urgently needed. We shed light on a previously unrecognized mechanism and unravel a key role of red blood cells, driving vascular dysfunction in patients with COVID-19 infection. We establish the presence of profound and persistent endothelial dysfunction in vivo in patients with COVID-19. Mechanistically, we show that targeting reactive oxygen species or arginase 1 improves vascular dysfunction mediated by red blood cells. These translational observations hold promise that restoring the redox balance in red blood cells might alleviate the clinical complications of COVID-19-associated vascular dysfunction.

Downregulation of Erythrocyte miR-210 Induces Endothelial Dysfunction in Type 2 Diabetes.

Red blood cells (RBC) act as mediators of vascular injury in type 2 diabetes mellitus (T2DM). miR-210 plays a protective role in cardiovascular homeostasis and is decreased in whole blood of T2DM mice. We hypothesized that downregulation of RBC miR-210 induces endothelial dysfunction in T2DM. RBC were coincubated with arteries and endothelial cells ex vivo and transfused in vivo to identify the role of miR-210 and its target protein tyrosine phosphatase 1B (PTP1B) in endothelial dysfunction. RBC from patients with T2DM and diabetic rodents induced endothelial dysfunction ex vivo and in vivo. miR-210 levels were lower in human RBC from patients with T2DM (T2DM RBC) than in RBC from healthy subjects. Transfection of miR-210 in human T2DM RBC rescued endothelial function, whereas miR-210 inhibition in healthy subjects RBC or RBC from miR-210 knockout mice impaired endothelial function. Human T2DM RBC decreased miR-210 expression in endothelial cells. miR-210 expression in carotid artery plaques was lower in T2DM patients than in patients without diabetes. Endothelial dysfunction induced by downregulated RBC miR-210 involved PTP1B and reactive oxygen species. miR-210 mimic attenuated endothelial dysfunction induced by RBC via downregulating vascular PTP1B and oxidative stress in diabetic mice in vivo. These data reveal that the downregulation of RBC miR-210 is a novel mechanism driving the development of endothelial dysfunction in T2DM.

MicroRNA: A mediator of diet-induced cardiovascular protection.

Diets containing nutrients such as polyunsaturated fatty acids, polyphenols, or vitamins have been shown to have cardiovascular benefits. Micro (mi)RNAs are fundamental regulators of gene expression and function in the cardiovascular system. Diet-induced cardiovascular benefits are associated with changes in endogenous expression of miRNAs in the cardiovascular system. In addition, emerging studies have shown that miRNAs present in the food can be transported in the circulation to tissues. These exogenous miRNAs may also affect cardiovascular function contributing to the diet-induced benefits. This review discusses the emerging role of both endogenous and exogenous miRNAs as mediators of diet-induced cardiovascular protection. Understanding the mechanisms of diet-mediated actions through modulation of miRNA may provide a potential strategy for new therapies.

Primary hypercholesterolemia and development of cardiovascular disorders: Cellular and molecular mechanisms involved in low-grade systemic inflammation and endothelial dysfunction.

Primary hypercholesterolemia, a metabolic disorder characterized by elevated circulating levels of cholesterol products, mainly low-density lipoproteins, is associated with arteriosclerosis development. Cardiovascular disease, predominantly myocardial infarction and stroke, remains the main cause of death worldwide, with atherosclerosis considered to be the most common underlying pathology. In addition to elevated plasma levels of low-density lipoproteins, low-grade systemic inflammation and endothelial dysfunction seem to be the main drivers of premature atherosclerosis. Here we review current knowledge related to cellular and molecular mechanisms involved in low-grade systemic inflammation and endothelial dysfunction associated with primary hypercholesterolemia. We also discuss the contribution of different inflammatory mediators, immune players and signaling pathways implicated in leukocyte adhesion to the dysfunctional endothelium, a key feature of atherogenesis development. A better understanding of these processes linked to primary hypercholesterolemia should shed new light on cardiovascular disease development and might guide novel and effective therapeutic strategies to impair its progression.

Oral Unsaturated Fat Load Impairs Postprandial Systemic Inflammation in Primary Hypercholesterolemia Patients.

Context: Primary hypercholesterolemia (PH) is a lipid disorder characterized by elevated levels of cholesterol and low-density lipoprotein (LDL). Low-grade systemic inflammation is associated with PH, which might explain the higher incidence of cardiovascular diseases in this setting. Objective: To evaluate the effect of an oral unsaturated fat load (OUFL) on different immune parameters and functional consequences in patients with PH in postprandial state. Design: A commercial liquid preparation of long-chain triglycerides (Supracal®; ω6/ω3 ratio >20/1, OUFL) was administered to 20 patients and 10 age-matched controls. Whole blood was collected before (fasting state) and 4 h after administration (postprandial state). Flow cytometry was employed to determine platelet and leukocyte activation, and the levels of circulating platelet-leukocyte aggregates. Soluble markers were determined by ELISA, and the parallel-plate flow chamber was employed to study leukocyte adhesion to the dysfunctional arterial endothelium. Results: The PH group had a lower percentage of activated platelets and circulating type 1 monocytes, and blunted neutrophil activation after the OUFL, accompanied by a significant increase in the percentage of regulatory T lymphocytes. In this group, the OUFL led to a significant impairment of leukocyte adhesion to the dysfunctional [tumor necrosis factor α (TNFα)-stimulated] endothelium and reduced the plasma levels of soluble P-selectin, platelet factor-4 (PF-4)/CXCL4, CXCL8, CCL2, CCL5, and TNFα. Conclusion: The OUFL has a beneficial impact on the pro-thrombotic and pro-inflammatory state of PH patients and might be a promising macronutrient approach to dampen the systemic inflammation associated with PH and the development of further cardiovascular events.

Systemic Inflammation in Metabolic Syndrome: Increased Platelet and Leukocyte Activation, and Key Role of CX3CL1/CX3CR1 and CCL2/CCR2 Axes in Arterial Platelet-Proinflammatory Monocyte Adhesion.

BACKGROUND: Metabolic syndrome is associated with low-grade systemic inflammation, which is a key driver of premature atherosclerosis. We characterized immune cell behavior in metabolic syndrome, its consequences, and the potential involvement of the CX3CL1/CX3CR1 and CCL2/CCR2 chemokine axes. METHODS: Whole blood from 18 patients with metabolic syndrome and 21 age-matched controls was analyzed by flow cytometry to determine the leukocyte immunophenotypes, activation, platelet-leukocyte aggregates, and CX3CR1 expression. ELISA determined the plasma marker levels. Platelet-leukocyte aggregates adhesion to tumor necrosis factor-α (TNFα)-stimulated arterial endothelium and the role of CX3CL1/CX3CR1 and CCL2/CCR2 axes was investigated with the parallel-plate flow chamber. RESULTS: When compared with the controls, the metabolic syndrome patients presented greater percentages of eosinophils, CD3+ T lymphocytes, Mon2/Mon3 monocytes, platelet-eosinophil and -lymphocyte aggregates, activated platelets, neutrophils, eosinophils, monocytes, and CD8+ T cells, but lower percentages of Mon1 monocytes. Patients had increased circulating interleukin-8 (IL-8) and TNFα levels and decreased IL-4. CX3CR1 up-regulation in platelet-Mon1 monocyte aggregates in metabolic syndrome patients led to increased CX3CR1/CCR2-dependent platelet-Mon1 monocyte adhesion to dysfunctional arterial endothelium. CONCLUSION: We provide evidence of generalized immune activation in metabolic syndrome. Additionally, CX3CL1/CX3CR1 or CCL2/CCR2 axes are potential candidates for therapeutic intervention in cardiovascular disorders in metabolic syndrome patients, as their blockade impairs the augmented arterial platelet-Mon1 monocyte aggregate adhesiveness, which is a key event in atherogenesis.

Genome-Wide Inhibition of Pro-atherogenic Gene Expression by Multi-STAT Targeting Compounds as a Novel Treatment Strategy of CVDs.

Cardiovascular diseases (CVDs), including atherosclerosis, are globally the leading cause of death. Key factors contributing to onset and progression of atherosclerosis include the pro-inflammatory cytokines Interferon (IFN)α and IFNγ and the Pattern Recognition Receptor (PRR) Toll-like receptor 4 (TLR4). Together, they trigger activation of Signal Transducer and Activator of Transcription (STAT)s. Searches for compounds targeting the pTyr-SH2 interaction area of STAT3, yielded many small molecules, including STATTIC and STX-0119. However, many of these inhibitors do not seem STAT3-specific. We hypothesized that multi-STAT-inhibitors that simultaneously block STAT1, STAT2, and STAT3 activity and pro-inflammatory target gene expression may be a promising strategy to treat CVDs. Using comparative in silico docking of multiple STAT-SH2 models on multi-million compound libraries, we identified the novel multi-STAT inhibitor, C01L_F03. This compound targets the SH2 domain of STAT1, STAT2, and STAT3 with the same affinity and simultaneously blocks their activity and expression of multiple STAT-target genes in HMECs in response to IFNα. The same in silico and in vitro multi-STAT inhibiting capacity was shown for STATTIC and STX-0119. Moreover, C01L_F03, STATTIC and STX-0119 were also able to affect genome-wide interactions between IFNγ and TLR4 by commonly inhibiting pro-inflammatory and pro-atherogenic gene expression directed by cooperative involvement of STATs with IRFs and/or NF-κB. Moreover, we observed that multi-STAT inhibitors could be used to inhibit IFNγ+LPS-induced HMECs migration, leukocyte adhesion to ECs as well as impairment of mesenteric artery contractility. Together, this implicates that application of a multi-STAT inhibitory strategy could provide great promise for the treatment of CVDs.

Novel Immune Features of the Systemic Inflammation Associated with Primary Hypercholesterolemia: Changes in Cytokine/Chemokine Profile, Increased Platelet and Leukocyte Activation.

Primary hypercholesterolemia (PH) is associated with a low grade systemic inflammation that is likely the main driver of premature atherosclerosis. Accordingly, we characterized the immune cell behaviour in PH and its potential consequences. Whole blood from 22 PH patients and 21 age-matched controls was analysed by flow cytometry to determine the percentage of leukocyte immunophenotypes, activation, and platelet-leukocyte aggregates. Plasma markers were determined by Enzyme-Linked ImmunoSorbent Assay (ELISA). The adhesion of platelet-leukocyte aggregates to tumor necrosis factor-α (TNFα)-stimulated arterial endothelium was investigated using the dynamic model of the parallel-plate flow chamber. PH patients presented greater percentage of Mon 3 monocytes, Th2 and Th17 lymphocytes, activated platelets, and leukocytes than controls. The higher percentages of circulating platelet-neutrophil, monocyte and lymphocyte aggregates in patients caused increased platelet-leukocyte adhesion to dysfunctional arterial endothelium. Circulating CXCL8, CCL2, CX3CL1, and IL-6 levels positively correlated with key lipid features of PH, whereas negative correlations were found for IL-4 and IL-10. We provide the first evidence that increased platelet and leukocyte activation leads to elevated platelet-leukocyte aggregates in PH and augmented arterial leukocyte adhesiveness, a key event in atherogenesis. Accordingly, modulation of immune system behavior might be a powerful target in the control of further cardiovascular disease in PH.

Functional role of endothelial CXCL16/CXCR6-platelet-leucocyte axis in angiotensin II-associated metabolic disorders.

Aims: Angiotensin-II (Ang-II) is the main effector peptide of the renin-angiotensin system (RAS) and promotes leucocyte adhesion to the stimulated endothelium. Because RAS activation and Ang-II signalling are implicated in metabolic syndrome (MS) and abdominal aortic aneurysm (AAA), we investigated the effect of Ang-II on CXCL16 arterial expression, the underlying mechanisms, and the functional role of the CXCL16/CXCR6 axis in these cardiometabolic disorders. Methods and results: Results from in vitro chamber assays revealed that CXCL16 neutralization significantly inhibited mononuclear leucocyte adhesion to arterial but not to venous endothelial cells. Flow cytometry and immunofluorescence studies confirmed that Ang-II induced enhanced endothelial CXCL16 expression, which was dependent on Nox5 up-regulation and subsequent RhoA/p38-MAPK/NFκB activation. Flow cytometry analysis further showed that MS patients had higher levels of platelet activation and a higher percentage of circulating CXCR6-expressing platelets, CXCR6-expressing-platelet-bound neutrophils, monocytes, and CD8+ lymphocytes than age-matched controls, leading to enhanced CXCR6/CXCL16-dependent adhesion to the dysfunctional (Ang-II- and TNFα-stimulated) arterial endothelium. Ang-II-challenged apolipoprotein E-deficient (apoE-/-) mice had a higher incidence of AAA, macrophage, CD3+, and CXCR6+ cell infiltration and neovascularization than unchallenged animals, which was accompanied by greater CCL2, CXCL16, and VEGF mRNA expression within the lesion together with elevated levels of circulating soluble CXCL16. Significant reductions in these parameters were found in animals co-treated with the AT1 receptor antagonist losartan or in apoE-/- mice lacking functional CXCR6 receptor (CXCR6GFP/GFP). Conclusion: CXCR6 expression on platelet-bound monocytes and CD8+ lymphocytes may constitute a new membrane-associated biomarker for adverse cardiovascular events. Moreover, pharmacological modulation of this axis may positively affect cardiovascular outcome in metabolic disorders linked to Ang-II.

Cigarette Smoke Increases Endothelial CXCL16-Leukocyte CXCR6 Adhesion In Vitro and In Vivo. Potential Consequences in Chronic Obstructive Pulmonary Disease.

Cardiovascular disease (CVD) is a major comorbidity in chronic obstructive pulmonary disease (COPD). Although the mechanism of its development remains largely unknown, it appears to be associated with cigarette consumption and reduced lung function. Therefore, the aim of this study was to investigate the potential link between water-soluble cigarette smoke extract (CSE)-induced endothelial dysfunction and the function of CXCL16/CXCR6 axis on the initial attachment of leukocytes, in addition to its possible impact on COPD-associated systemic inflammation. To do this, we employed several experimental approaches, including RNA silencing and flow cytometry analysis, the dynamic flow chamber technique, and intravital microscopy in the cremasteric arterioles of animals exposed to cigarette smoke (CS). CSE-induced arterial CXCL16 expression, leading to increased platelet-leukocyte and mononuclear cell adhesiveness. CSE-induced CXCL16 expression was dependent on Nox5 expression and subsequent RhoA/p38 MAPK/NF-κB activation. Flow cytometry analysis revealed that COPD patients (n = 35) presented greater numbers of activated circulating platelets (PAC-1+ and P-selectin+) expressing CXCL16 and CXCR6 as compared with age-matched controls (n = 17), with a higher number of CXCR6+-platelets in the smoking COPD group than in ex-smokers. This correlated with enhanced circulating CXCR6+-platelet-leukocyte aggregates in COPD patients. The increase in circulating numbers of CXCR6-expressing platelets and mononuclear cells resulted in enhanced platelet-leukocyte and leukocyte adhesiveness to CSE-stimulated arterial endothelium, which was greater than that found in age-matched controls and was partly dependent on endothelial CXCL16 upregulation. Furthermore, CS exposure provoked CXCL16-dependent leukocyte-arteriolar adhesion in cremasteric arterioles, which was significantly reduced in animals with a nonfunctional CXCR6 receptor. In conclusion, we provide the first evidence that increased numbers of CXCR6-expressing circulating platelets and mononuclear leukocytes from patients with COPD might be a marker of systemic inflammation with potential consequences in CVD development. Accordingly, CXCL16/CXCR6 axis blockade might constitute a new therapeutic approach for decreasing the risk of CVD in COPD patients.