Mechanisms of endothelial activation, hypercoagulation and thrombosis in COVID-19: a link with diabetes mellitus.

Early since the onset of the COVID-19 pandemic, the medical and scientific community were aware of extra respiratory actions of SARS-CoV-2 infection. Endothelitis, hypercoagulation, and hypofibrinolysis were identified in COVID-19 patients as subsequent responses of endothelial dysfunction. Activation of the endothelial barrier may increase the severity of the disease and contribute to long-COVID syndrome and post-COVID sequelae. Besides, it may cause alterations in primary, secondary, and tertiary hemostasis. Importantly, these responses have been highly decisive in the evolution of infected patients also diagnosed with diabetes mellitus (DM), who showed previous endothelial dysfunction. In this review, we provide an overview of the potential triggers of endothelial activation related to COVID-19 and COVID-19 under diabetic milieu. Several mechanisms are induced by both the viral particle itself and by the subsequent immune-defensive response (i.e., NF-κB/NLRP3 inflammasome pathway, vasoactive peptides, cytokine storm, NETosis, activation of the complement system). Alterations in coagulation mediators such as factor VIII, fibrin, tissue factor, the von Willebrand factor: ADAMST-13 ratio, and the kallikrein-kinin or plasminogen-plasmin systems have been reported. Moreover, an imbalance of thrombotic and thrombolytic (tPA, PAI-I, fibrinogen) factors favors hypercoagulation and hypofibrinolysis. In the context of DM, these mechanisms can be exacerbated leading to higher loss of hemostasis. However, a series of therapeutic strategies targeting the activated endothelium such as specific antibodies or inhibitors against thrombin, key cytokines, factor X, complement system, the kallikrein-kinin system or NETosis, might represent new opportunities to address this hypercoagulable state present in COVID-19 and DM. Antidiabetics may also ameliorate endothelial dysfunction, inflammation, and platelet aggregation. By improving the microvascular pathology in COVID-19 and post-COVID subjects, the associated comorbidities and the risk of mortality could be reduced.

Redox Regulation of Microglial Inflammatory Response: Fine Control of NLRP3 Inflammasome through Nrf2 and NOX4.

The role of inflammation and immunity in the pathomechanism of neurodegenerative diseases has become increasingly relevant within the past few years. In this context, the NOD-like receptor protein 3 (NLRP3) inflammasome plays a crucial role in the activation of inflammatory responses by promoting the maturation and secretion of pro-inflammatory cytokines such as interleukin-1ß and interleukin-18. We hypothesized that the interplay between nuclear factor erythroid 2-related factor 2 (Nrf2) and NADPH oxidase 4 (NOX4) may play a critical role in the activation of the NLRP3 inflammasome and subsequent inflammatory responses. After priming mixed glial cultures with lipopolysaccharide (LPS), cells were stimulated with ATP, showing a significant reduction of IL1-ß release in NOX4 and Nrf2 KO mice. Importantly, NOX4 inhibition using GKT136901 also reduced IL-1ß release, as in NOX4 KO mixed glial cultures. Moreover, we measured NOX4 and NLRP3 expression in wild-type mixed glial cultures following LPS treatment, observing that both increased after TLR4 activation, while 24 h treatment with tert-butylhydroquinone, a potent Nrf2 inducer, significantly reduced NLRP3 expression. LPS administration resulted in significant cognitive impairment compared to the control group. Indeed, LPS also modified the expression of NLRP3 and NOX4 in mouse hippocampus. However, mice treated with GKT136901 after LPS impairment showed a significantly improved discrimination index and recovered the expression of inflammatory genes to normal levels compared with wild-type animals. Hence, we here validate NOX4 as a key player in NLRP3 inflammasome activation, suggesting NOX4 pharmacological inhibition as a potent therapeutic approach in neurodegenerative diseases.

Resolvin E1 attenuates doxorubicin-induced endothelial senescence by modulating NLRP3 inflammasome activation.

Endothelial cell senescence contributes to chronic inflammation and endothelial dysfunction, while favoring cardiovascular disorders and frailty. Senescent cells acquire a pro-inflammatory secretory phenotype that further propagates inflammation and senescence to neighboring cells. Cell senescence can be provoked by plethora of stressors, including inflammatory molecules and chemotherapeutic drugs. Doxorubicin (Doxo) is a powerful anthracycline anticancer drug whose clinical application is constrained by a dose-limiting cardiovascular toxicity. We here investigated whether cell senescence can contribute to the vascular damage elicited by Doxo. In human umbilical vein endothelial cells (HUVEC) cultures, Doxo (10-100 nM) increased the number of SA-ß-gal positive cells and the levels of γH2AX, p21 and p53, used as markers of senescence. Moreover, we identified Doxo-induced senescence to be mediated by the nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome, a key player of the immune innate system capable of releasing interleukin (IL)-1ß. In fact, IL-1ß itself mimicked the stimulatory action of Doxo on both NLRP3 activation and cellular senescence, while the pharmacological blockade of IL-1 receptors markedly attenuated the pro-senescence effects of Doxo. In search of additional pharmacological strategies to attenuate Doxo-induced endothelial senescence, we identified resolvin E1 (RvE1), an endogenous pro-resolving mediator, as capable of reducing cell senescence induced by both Doxo and IL-1ß by interfering with the increased expression of pP65, NLRP3, and pro-IL-1ß proteins and with the formation of active NLRP3 inflammasome complexes. Overall, RvE1 and the blockade of the NLRP3 inflammasome-IL-1ß axis may offer a novel therapeutic approach against Doxo-induced cardiovascular toxicity and subsequent sequelae.

DPP4 Promotes Human Endothelial Cell Senescence and Dysfunction via the PAR2-COX-2-TP Axis and NLRP3 Inflammasome Activation.

BACKGROUND: Abnormal accumulation of senescent cells in the vessel wall leads to a compromised vascular function contributing to vascular aging. Soluble DPP4 (dipeptidyl peptidase 4; sDPP4) secretion from visceral adipose tissue is enhanced in obesity, now considered a progeric condition. sDPP4 triggers vascular deleterious effects, albeit its contribution to vascular aging is unknown. We aimed to explore sDPP4 involvement in vascular aging, unraveling the molecular pathway by which sDPP4 acts on the endothelium. METHODS: Human endothelial cell senescence was assessed by senescence-associated ß-galactosidase assay, visualization of DNA damage, and expression of prosenescent markers, whereas vascular function was evaluated by myography over human dissected microvessels. In visceral adipose tissue biopsies from a cohort of obese patients, we explored several age-related parameters in vitro and ex vivo. RESULTS: By a common mechanism, sDPP4 triggers endothelial cell senescence and endothelial dysfunction in isolated human resistance arteries. sDPP4 activates the metabotropic receptor PAR2 (protease-activated receptor 2), COX-2 (cyclooxygenase 2) activity, and the production of TXA2 (thromboxane A2) acting over TP (thromboxane receptor) receptors (PAR2-COX-2-TP axis), leading to NLRP3 (nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing 3) inflammasome activation. Obese patients exhibited impaired microarterial functionality in comparison to control nonobese counterparts. Importantly, endothelial dysfunction in obese patients positively correlated with greater expression of DPP4, prosenescent, and proinflammatory markers in visceral adipose tissue nearby the resistance arteries. Moreover, when DPP4 activity or sDPP4-induced prosenescent mechanism was blocked, endothelial dysfunction was restored back to levels of healthy subjects. CONCLUSIONS: These results reveal sDPP4 as a relevant mediator in early vascular aging and highlight its capacity activating main proinflammatory mediators in the endothelium that might be pharmacologically tackled.

NLRP3 Inflammasome in Vascular Disease: A Recurrent Villain to Combat Pharmacologically.

Despite the great advances in medicine, mortality from cardiovascular diseases keeps on growing. This tendency is not likely to change considering the pandemic proportions of obesity and diabetes. Besides, the global population is more aged as life expectancy increases, and vascular aging plays a key role in the increased risk of vascular disease. In light of recent trials, namely the CANTOS study, showing the enormous potential of anti-inflammatory therapies and in particular those targeted to IL-1ß, a change in therapeutical management of cardiovascular diseases is coming about. The NLRP3 inflammasome is a multiprotein complex that assembles to engage the innate immune defense by processing the maturation of pro-inflammatory cytokines IL-1ß and IL-18. Substantial evidence has positioned the NLRP3 inflammasome at the center of vascular disease progression, with a particular significance in the context of aging and the low-grade chronic inflammation associated (inflammaging). Therefore, pharmacological blockade of the NLRP3 inflammasome and its end products has arisen as an extremely promising tool to battle vascular disease. In this review, we discuss the mechanisms by which the NLRP3 inflammasome contributes to vascular disease, with particular attention to the consequences of aging, and we enumerate the therapeutic options available to combat this recurrent villain.

Pharmacological Blockade of NLRP3 Inflammasome/IL-1ß-Positive Loop Mitigates Endothelial Cell Senescence and Dysfunction.

The clinical relevance of IL-1ß in chronic inflammation underlying atherosclerosis has been reinforced by recent evidence associating pharmacological inhibition of the cytokine with lower cardiovascular risk. Previously, we have demonstrated a direct involvement of IL-1ß in endothelial senescence. Therefore, this can be a key mechanism contributing to the sterile inflammatory milieu associated with aging, termed inflammaging. In the present study, we have evaluated whether a positive feedback of IL-1ß in the NLRP3 inflammasome via NF-κB could promote human endothelial senescence in vitro and murine endothelial dysfunction in vivo. Our results indicate that the NLRP3 inflammasome is pivotal in mediating the detrimental effects of IL-1ß, showing that auto-activation is a crucial feature boosting endothelial cell senescence in vitro, which is paralleled by vascular dysfunction in vivo. Hence, the inhibitor of NLRP3 inflammasome assembly, MCC 950, was able to disrupt the aforementioned positive loop, thus alleviating inflammation, cell senescence and vascular dysfunction. Besides, we explored alternative NLRP3 inflammasome inhibitory agents such as the RAS heptapeptide Ang-(1-7) and the anti-aging protein klotho, both of which demonstrated protective effects in vitro and in vivo. Altogether, our results highlight a fundamental role for the hereby described NLRP3 inflammasome/IL-1ß positive feedback loop in stress-induced inflammaging and the associated vascular dysfunction, additionally providing evidence of a potential therapeutic use of MCC 950, Ang-(1-7) and recombinant klotho to block this loop and its deleterious effects.

DPP4 and ACE2 in Diabetes and COVID-19: Therapeutic Targets for Cardiovascular Complications?

COVID-19 outbreak, caused by severe acute respiratory syndrome (SARS)-CoV-2 coronavirus has become an urgent health and economic challenge. Diabetes is a risk factor for severity and mortality of COVID-19. Recent studies support that COVID-19 has effects beyond the respiratory tract, with vascular complications arising as relevant factors worsening its prognosis, then making patients with previous vascular disease more prone to severity or fatal outcome. Angiotensin-II converting enzime-2 (ACE2) has been proposed as preferred receptor for SARS-CoV-2 host infection, yet specific proteins participating in the virus entry are not fully known. SARS-CoV-2 might use other co-receptor or auxiliary proteins allowing virus infection. In silico experiments proposed that SARS-CoV-2 might bind dipeptidyl peptidase 4 (DPP4/CD26), which was established previously as receptor for MERS-CoV. The renin-angiotensin-aldosterone system (RAAS) component ACE2 and DPP4 are proteins dysregulated in diabetes. Imbalance of the RAAS and direct effect of soluble DPP4 exert deleterious vascular effects. We hypothesize that diabetic patients might be more affected by COVID-19 due to increased presence ACE2 and DPP4 mediating infection and contributing to a compromised vasculature. Here, we discuss the role of ACE2 and DPP4 as relevant factors linking the risk of SARS-CoV-2 infection and severity of COVID-19 in diabetic patients and present an outlook on therapeutic potential of current drugs targeted against RAAS and DPP4 to treat or prevent COVID-19-derived vascular complications. Diabetes affects more than 400 million people worldwide, thus better understanding of how they are affected by COVID-19 holds an important benefit to fight against this disease with pandemic proportions.

Visfatin/eNampt induces endothelial dysfunction in vivo: a role for Toll-Like Receptor 4 and NLRP3 inflammasome.

Visfatin/extracellular-nicotinamide-phosphoribosyltranferase-(eNampt) is a multifaceted adipokine enhanced in type-2-diabetes and obesity. Visfatin/eNampt cause in vitro endothelial dysfunction and vascular inflammation, although whether the same effects are achieved in vivo is unknown. Toll-like receptor-4 (TLR4), a main surface pattern recognition receptor of innate immune system is a potential target for visfatin/eNampt. We studied its capacity to generate vascular dysfunction in vivo, focusing on TLR4 role and downstream activation of nod-like-receptor-protein-3 (NLRP3)-inflammasome. 4 month-old C57BL/6 mice were exposed to 7 days infusion of visfatin/eNampt, alone or together with FK 866 (Nampt enzymatic inhibitor), CLI 095 (TLR4 blocker), MCC 950 (NLRP3-inflammasome inhibitor), or anakinra (interleukin(IL)-1-receptor antagonist). Endothelial dysfunction was tested in isolated microvessels. In human umbilical endothelial cells (HUVEC), proteins related to the NLRP3-inflammasome phosphorylated p-65, NLRP3, caspase-1, pro-IL-1ß, and mature IL-1ß were determined by Western blot, while the inflammasome related apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC-specks) was studied by immunofluorescence. Impaired endothelium-dependent relaxations were observed in isolated mesenteric microvessels from visfatin/eNampt-infused mice. This effect was attenuated by co-treatment with FK 866 or CLI 095, supporting a role for Nampt enzymatic activity and TLR4 activation. Moreover, cultured HUVEC exposed to visfatin/eNampt showed higher expression and activation of NLRP3-inflammasome. Again, this effect relied on Nampt enzymatic activity and TLR4 activation, and it was abrogated by the inflammasome assembly blockade with MCC 950. The endothelial dysfunction evoked by visfatin/eNampt infusion in vivo was also sensitive to both MCC 950 and anakinra treatments, suggesting that the NLRP3-inflammasome-driven tissular release of IL-1ß is the final mediator of endothelial damage. We conclude that Visfatin/eNampt produces in vivo vascular dysfunction in mice by a Nampt-dependent TLR4-mediated pathway, involving NLRP3-inflammasome and paracrine IL-1ß. Thus, those targets may become therapeutic strategies for attenuating the adipokine-mediated vascular dysfunction associated to obesity and/or type-2-diabetes.

The angiotensin-(1-7)/Mas receptor axis protects from endothelial cell senescence via klotho and Nrf2 activation.

Endothelial cell senescence is a hallmark of vascular aging that predisposes to vascular disease. We aimed to explore the capacity of the renin-angiotensin system (RAS) heptapeptide angiotensin (Ang)-(1-7) to counteract human endothelial cell senescence and to identify intracellular pathways mediating its potential protective action. In human umbilical vein endothelial cell (HUVEC) cultures, Ang II promoted cell senescence, as revealed by the enhancement in senescence-associated galactosidase (SA-ß-gal+) positive staining, total and telomeric DNA damage, adhesion molecule expression, and human mononuclear adhesion to HUVEC monolayers. By activating the G protein-coupled receptor Mas, Ang-(1-7) inhibited the pro-senescence action of Ang II, but also of a non-RAS stressor such as the cytokine IL-1ß. Moreover, Ang-(1-7) enhanced endothelial klotho levels, while klotho silencing resulted in the loss of the anti-senescence action of the heptapeptide. Indeed, both Ang-(1-7) and recombinant klotho activated the cytoprotective Nrf2/heme oxygenase-1 (HO-1) pathway. The HO-1 inhibitor tin protoporphyrin IX prevented the anti-senescence action evoked by Ang-(1-7) or recombinant klotho. Overall, the present study identifies Ang-(1-7) as an anti-senescence peptide displaying its protective action beyond the RAS by consecutively activating klotho and Nrf2/HO-1. Ang-(1-7) mimetic drugs may thus prove useful to prevent endothelial cell senescence and its related vascular complications.