New insights into the role and therapeutic potential of HSP70 in diabetes.

Emerging evidence indicates that HSP70 represents a key mechanism in the pathophysiology of ß-cell dysfunction, insulin resistance, and various diabetic complications, including micro- and macro-vascular alterations, as well as impaired hemostasis. Hyperglycemia, a hallmark of both types of diabetes, increases the circulating levels of HSP70 (eHSP70), but there is still divergence about whether diabetes up- or down-regulates the intracellular fraction of this protein (iHSP70). Here, we consider that iHSP70 levels reduce in diabetic arterial structures and that the vascular system is in direct contact with all other systems in the body suggesting that a systemic response might also be happening for iHSP70, which is characterized by decreased levels of HSP70 in the vasculature. Furthermore, although many pathways have been proposed to explain HSP70's functions in diabetes, and organs/tissues/cells-specific variations occur, the membrane-bound receptor of the innate immune system, Toll-like receptor 4, and its downstream signal transduction pathways appear to be a constant, not only when we explore the actions of eHSP70, but also when we assess the contributions of iHSP70. In this review, we focus on discussing the multiple roles of HSP70 across organs/tissues/cells affected by hyperglycemia to further explore the possibility of targeting this protein with pharmacological and non-pharmacological approaches in the context of diabetes.

PnPP-19 Peptide Restores Erectile Function in Hypertensive and Diabetic Animals Through Intravenous and Topical Administration.

INTRODUCTION: With the aim of overcoming the high toxicity of PnTx2-6 (or δ-CNTX-Pn2a), a toxin from the venom of the armed spider (Phoneutria nigriventer), the 19-aminoacid peptide, PnPP-19 (P nigriventer potentiator peptide), was synthesized based on molecular modeling studies of PnTx2-6. PnPP-19 improved the erectile function of normotensive rats and mice, without eliciting side effects, and no signs of toxicity were observed. In addition, PnPP-19 was able to potentiate the effect of sildenafil. AIM: To evaluate the efficacy of PnPP-19 in hypertensive and diabetic mouse/rat models in restoring erectile function, after topical administration; verify the biodistribution of PnPP-19 administration (topical and intravenous), permeation, and cyclic guanosine monophosphate (cGMP)/nitric oxide via implication. METHODS: Corpus cavernosum relaxation was evaluated using cavernous strips from male spontaneous hypertensive rats (SHR) and from streptozotocin (STZ)-diabetic mice contracted with phenylephrine and submitted to electrical field stimulation before and after incubation with PnPP-19 (10-8 mol/L, 10 minutes) or vehicle. This procedure was also used to determine cGMP/nitric oxide levels, at 8 Hz and to check the effect of PnPP-19 with sildenafil citrate. Biodistribution assays were performed using iodine 123-radiolabeled PnPP-19. In vivo erectile function was evaluated using intracavernosal pressure/main arterial pressure ratio in STZ-diabetic rats after PnPP-19 topical administration. MAIN OUTCOME MEASURES: PnPP-19 may become a new drug able to fill the gap in the pharmacologic treatment of erectile dysfunction, especially for hypertensive and diabetic individuals RESULTS: PnPP-19 potentiated corpus cavernosum relaxation, in both control and SHR rats. SHR-cavernosal tissue treated with PnPP-19 (1-32 Hz) reached the same relaxation levels as control Wistar rats (16 and 32 Hz). PnPP-19 treatment improved cavernosal tissue relaxation in STZ-diabetic mice and rats. PnPP-19 enhanced cGMP levels in STZ-diabetic mice corpus cavernosum strips. After topical or intravenous administration in rats, 123I-PnPP-19 was mainly recruited to the penis. When topically administered (400 µg/rat), PnPP-19 restores erectile function in STZ-diabetic rats, also improving it in healthy rats by increasing the intracavernosal pressure/main arterial pressure ratio. PnPP-19 exhibited an additive effect when co-administered with sildenafil, showing a novel mode of action regardless of phosphodiesterase type 5 inhibition. CLINICAL IMPLICATIONS: PnPP-19 seems to be an indicated drug to be tested to treat ED in diabetic and hypertensive patients. STRENGTH & LIMITATIONS: PnPP-19, although active by topical application and showing safety to human beings (not shown), has low permeability, about 10% of the applied dose. CONCLUSION: Our results showed that PnPP-19 may emerge as a potent new drug that can be topically administered, becoming a promising alternative for erectile dysfunction treatment. Nunes da Silva C, Pedrosa Nunes K, De Marco Almeida F, et al. PnPP-19 Peptide Restores Erectile Function In Hypertensive And Diabetic Animals Through Intravenous And Topical Administration. J Sex Med 2019;16:365-374.

Unveiling the Interplay between the TLR4/MD2 Complex and HSP70 in the Human Cardiovascular System: A Computational Approach.

While precise mechanisms underlying cardiovascular diseases (CVDs) are still not fully understood, previous studies suggest that the innate immune system, through Toll-like receptor 4 (TLR4), plays a crucial part in the pathways leading to these diseases, mainly because of its interplay with endogenous molecules. The Heat-shock protein 70 family (HSP70-70kDa) is of particular interest in cardiovascular tissues as it may have dual effects when interacting with TLR4 pathways. Although the hypothesis of the HSP70 family members acting as TLR4 ligands is becoming widely accepted, to date no co-crystal structure of this complex is available and it is still unknown whether this process requires the co-adaptor MD2. In this study, we aimed at investigating the interplay between the TLR4/MD2 complex and HSP70 family members in the human cardiovascular system through transcriptomic data analysis and at proposing a putative interaction model between these proteins. We report compelling evidence of correlated expression levels between TLR4 and MD2 with HSP70 cognate family members, especially in heart tissue. In our molecular docking simulations, we found that HSP70 in the ATP-bound state presents a better docking score towards the TLR4/MD2 complex compared to the ADP-bound state (-22.60 vs. -10.29 kcal/mol, respectively). Additionally, we show via a proximity ligation assay for HSP70 and TLR4, that cells stimulated with ATP have higher formation of fluorescent spots and that MD2 might be required for the complexation of these proteins. The insights provided by our computational approach are potential scaffolds for future in vivo studies investigating the interplay between the TLR4/MD2 complex and HSP70 family members in the cardiovascular system.

Blockade of Toll-Like Receptor 4 Attenuates Erectile Dysfunction in Diabetic Rats.

INTRODUCTION: While increased toll-like receptor (TLR)4 activity may contribute to the pathophysiology of vascular diseases, the molecular mechanisms disrupted by this receptor in the vasculature are still poorly understood. Additionally, it is unknown if TLR4 mediates erectile dysfunction (ED) during diabetes. AIM: To investigate whether pharmacological blockade of TLR4 affects erectile function in a murine model of diabetes. METHODS: Sprague Dawley rats (Charles River Laboratory, Wilmington, MA, USA) received a single streptozotocin injection (65 mg/kg, 28 days) and were treated with an anti-TLR4 antibody (1 µg/d, intraperitoneally) for the last 14 days of the treatment. Additionally, cavernosal strips were acutely incubated for 30 minutes with CLI-095 (10-5 mol/L), a TLR4 inhibitor. Functional studies, Western blotting, erectile function, immunohistochemistry, and biochemical analyses were performed. MAIN OUTCOME MEASURES: Oxidative stress, cyclic guanosine monophosphate (cGMP) levels, and functional studies were evaluated in treated and nontreated cavernosal strips from control and diabetic animals. Additionally, in vivo erectile function was assessed. RESULTS: Enhanced TLR4 expression was observed in corpus cavernosum from diabetic rats compared with control animals. Long-term blockade of TLR4 slightly improved diabetes-induced ED in rats due to attenuation of oxidative stress and increased cGMP levels in penile tissue, which ameliorated cavernosal relaxation. Functional experiments revealed that acute or chronic inhibition of TLR4 decreased hypercontractility in response to phenylephrine and improved nitrergic relaxation in corpus cavernosum from diabetic rats. CLINICAL IMPLICATIONS: TLR4 blockade may be a novel therapeutic strategy to assist in ED management. STRENGTHS & LIMITATIONS: The strength of this article stems from the fact that we showed that TLR4 blockade partly improves erectile function in vivo in diabetic rats. Its limitations mainly include that messenger RNA analysis for the nitric oxide/cGMP pathway were not performed. CONCLUSION: In summary, TLR4 participates in the mechanisms of diabetes-associated ED and blockade of this receptor positively affects penile vascular function. Nunes KP, de Oliveira AA, Szasz T, et al. Blockade of Toll-Like Receptor 4 Attenuates Erectile Dysfunction in Diabetic Rats. J Sex Med 2018;15:1235-1245.

The interplay between Angiotensin II, TLR4 and hypertension.

Hypertension is a multifactorial disease. Although a number of different underlying mechanisms have been learned from the various experimental models of the disease, hypertension still poses challenges for treatment. Angiotensin II plays an unquestionable role in blood pressure regulation acting through central and peripheral mechanisms. During hypertension, dysregulation of the Renin-Angiotensin System is associated with increased expression of pro-inflammatory cytokines and reactive oxygen species causing kidney damage, endothelial dysfunction, and increase in sympathetic activity, among other damages, eventually leading to decline in organ function. Recent studies have shown that these effects involve both the innate and the adaptive immune response. The contribution of adaptive immune responses involving different lymphocyte populations in various models of hypertension has been extensively studied. However, the involvement of the innate immunity mediating inflammation in hypertension is still not well understood. The innate and adaptive immune systems intimately interact with one another and are essential to an effectively functioning of the immune response; hence, the importance of a better understanding of the underlying mechanisms mediating innate immune system during hypertension. In this review, we aim to discuss mechanisms linking Angiotensin II and the innate immune system, in the pathogenesis of hypertension. The newest research investigating Angiotensin II triggering toll like receptor 4 activation in the kidney, vasculature and central nervous system contributing to hypertension will be discussed. Understanding the role of the innate immune system in the development of hypertension may bring to light new insights necessary to improve hypertension management.

Beneficial effect of the soluble guanylyl cyclase stimulator BAY 41-2272 on impaired penile erection in db/db-/- type II diabetic and obese mice.

Type 2 diabetes mellitus (DM2) and obesity are major risk factors for erectile dysfunction (ED). In diabetes, increased oxidative stress leads to decreased nitric oxide (NO) bioavailability, and diabetic patients appear to be less responsive to conventional therapy with phosphodiesterase type 5 inhibitors. We investigated whether the soluble guanylyl cyclase stimulator BAY 41-2272 (5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]pyrimidin-4ylamine) is effective in improving impaired corpus cavernosum (CC) relaxation in obese DM2 mice by reducing oxidative stress. Adult db/db(-/-) mice or their lean db(/+) littermates were used to assess vascular function, cGMP levels, antioxidant status, NADPH oxidase expression, and superoxide formation in the absence or presence of BAY 41-2272. Results showed that BAY 41-2272 (10(-8) to 10(-5) M) potently relaxed CC from db(/+) or db/db(-/-) mice in a similar manner. BAY 41-2272 significantly enhanced both endothelium-dependent and nitrergic relaxation induced by electrical field stimulation (EFS), and improved the impaired relaxation to acetylcholine and EFS in the diabetic animals in a concentration-dependent manner (10(-8) to 10(-7) M). BAY 41-2272 increased cGMP levels and potentiated relaxation responses to exogenous NO in CC. Total antioxidant status was reduced in plasma and urine whereas expression of vascular NADPH oxidase subunits (gp91phox, p22phox, and p47phox) was increased in the CC of db/db(-/-) mice, suggesting a state of oxidative stress. These effects were prevented by BAY 41-2272 in a concentration-dependent manner. These results suggest that BAY 41-2272 improves CC relaxation in db/db(-/-) mice by increasing cGMP and augmenting antioxidant status, making this drug is a potential novel candidate to treat ED.

PnPP-19, a Synthetic and Nontoxic Peptide Designed from a Phoneutria nigriventer Toxin, Potentiates Erectile Function via NO/cGMP.

PURPOSE: We designed a peptide, PnPP-19, comprising the potential active core of the Phoneutria nigriventer native toxin PnTx2-6. We investigated its role on erectile function, and its toxicity and immunogenicity. MATERIALS AND METHODS: Erectile function was evaluated by the intracavernous pressure-to-mean arterial pressure ratio during electrical field stimulation on rat pelvic ganglia. Cavernous strips were contracted with phenylephrine and relaxation was induced by electrical field stimulation with or without PnPP-19 (10(-8) M). Activity on sodium channels was evaluated by electrophysiological screening of transfected channels on Xenopus oocytes and dorsal root ganglion cells. Antibodies were detected by indirect enzyme-linked immunosorbent assay in mice previously treated with the peptide. Histopathological studies were performed with mouse organs treated with different doses of PnPP-19. RESULTS: PnPP-19 was able to potentiate erection at 4 and 8 Hz in vivo and ex vivo. It showed no toxicity and low immunogenicity in mice, and did not affect sodium channels or rat hearts. PnPP-19 increased cyclic guanosine monophosphate levels at 8 Hz. This effect was inhibited by L-NAME (10(-4) M). Erectile function was partially inhibited by 7-nitroindazole (10(-5) M), a selective inhibitor of neuronal nitric oxide synthase. CONCLUSIONS: PnPP-19 potentiates erection in vivo and ex vivo via the nitric oxide/cyclic guanosine monophosphate pathway. It does not affect sodium channels or rat hearts and shows no toxicity and low immunogenicity. These findings make it a promising candidate as a novel drug in the therapy of erectile dysfunction.

HSP70: From Signaling Mechanisms to Therapeutics.

Heat-shock proteins (HSPs) are primary stress responders that are vital to maintaining homeostasis [...].

New insights into hypertension-associated erectile dysfunction.

PURPOSE OF REVIEW: Erectile dysfunction is recognized as a quality-of-life disorder that needs to be treated. Currently, it is estimated to affect as many as 30 million American men. Thirty percent of hypertensive patients complain of erectile dysfunction. The understanding of common mechanisms involved in the cause of erectile dysfunction associated with hypertension, and the investigation of antihypertensive drugs that impact erectile dysfunction, will provide important tools toward identifying new therapeutic targets that will improve the quality of life for patients in these conditions. RECENT FINDINGS: Hypertension and erectile dysfunction are closely intertwined diseases, which have endothelial dysfunction as a common base. During hypertension and/or erectile dysfunction, disturbance of endothelium-derived factors can lead to an increase in vascular smooth muscle (VSM) contraction. Hypertension can lead to erectile dysfunction as a consequence of high blood pressure (BP) or due to antihypertensive treatment. However, growing evidence suggests erectile dysfunction is an early sign for hypertension. Also, some phosphodiesterase-5 inhibitors used to treat erectile dysfunction can improve BP, but the link between these conditions has not been totally understood. SUMMARY: This review will discuss the interplay between hypertension and erectile dysfunction, exploring newest insights regarding hypertension-associated erectile dysfunction, as well as the effect of antihypertensive drugs in erectile dysfunction patients.

Increased eHSP70-to-iHSP70 ratio disrupts vascular responses to calcium and activates the TLR4-MD2 complex in type 1 diabetes.

AIMS: Vascular dysfunction is a clinical hallmark of diabetes. While various pathways drive vascular alterations in diabetes, many gaps persist in understanding this process. Heat-shock protein 70 (HSP70) has a long-recognized role in diabetes, but the contributions of HSP70 to the diabetic vasculature remain largely unknown. MAIN METHODS: We determined the systemic and local (aorta) levels of HSP70 in control (CTL) and streptozotocin (STZ)-induced diabetic rats. Functional studies were conducted in a wire myograph in the presence or absence of a pharmacological inhibitor for HSP70 (VER155008). Calcium (Ca2+) dynamics was indirectly evaluated as a function of change in force development in vehicle and VER-treated vessels, as well as in the presence of inhibitors for voltage-dependent and -independent plasmalemmal Ca2+ channels. Furthermore, mimicking the extracellular diabetic environment, we exposed aortic rings to serum from CTL and STZ-induced animals, which contains higher levels of HSP70, as well as to purified recombinant HSP70. Then, we performed functional studies following the modulation of Toll-like receptor 4 (TLR4) and its co-adaptor MD2, which interact with HSP70. KEY FINDINGS: HSP70 plays a dual role in diabetes-induced vascular dysfunction: intracellular (i)HSP70 affects Ca2+ handling mechanisms, and extracellular (e)HSP70 modulates the TLR4-MD2 complex. SIGNIFICANCE: These newly discovered roles of HSP70 push forward the field of vascular biology and open research avenues for other diseased states associated with altered vascular responses.

From the PnTx2-6 Toxin to the PnPP-19 Engineered Peptide: Therapeutic Potential in Erectile Dysfunction, Nociception, and Glaucoma.

The venom of the "armed" spider Phoneutria nigriventer comprises several potent toxins. One of the most toxic components from this venom is the neurotoxin PnTx2-6 (LD50 = ∼ 0.7 µg/mouse, 48 residues, five disulfide bridges, MW = 5,289.31 Da), which slows down the inactivation of various Na+ channels. In mice and rats, this toxin causes priapism, an involuntary and painful erection, similar to what is observed in humans bitten by P. nigriventer. While not completely elucidated, it is clear that PnTx2-6 potentiates erectile function via NO/cGMP signaling, but it has many off-target effects. Seeking to obtain a simpler and less toxic molecule able to retain the pharmacological properties of this toxin, we designed and synthesized the peptide PnPP-19 (19 residues, MW = 2,485.6 Da), representing a discontinuous epitope of PnTx2-6. This synthetic peptide also potentiates erectile function via NO/cGMP, but it does not target Na+ channels, and therefore, it displays nontoxic properties in animals even at high doses. PnPP-19 effectively potentiates erectile function not only after subcutaneous or intravenous administration but also following topical application. Surprisingly, PnPP-19 showed central and peripheral antinociceptive activity involving the opioid and cannabinoid systems, suggesting applicability in nociception. Furthermore, considering that PnPP-19 increases NO availability in the corpus cavernosum, this peptide was also tested in a model of induced intraocular hypertension, characterized by low NO levels, and it showed promising results by decreasing the intraocular pressure which prevents retinal damage. Herein, we discuss how was engineered this smaller active non-toxic peptide with promising results in the treatment of erectile dysfunction, nociception, and glaucoma from the noxious PnTx2-6, as well as the pitfalls of this ongoing journey.

RhoA/Rho-kinase and vascular diseases: what is the link?

RhoA/Rho-kinase pathway plays an important role in many pathological conditions. RhoA participates in the regulation of smooth muscle tone and activates many downstream kinases. The best characterized are the serine/threonine kinase isoforms (Rho-kinase or ROCK), ROCKα/ROCK2 and ROCKß/ROCK1. ROCK is necessary for diverse functions such as local blood flow, arterial/pulmonary blood pressure, airway resistance and intestinal peristalsis. ROCK activation permits actin/myosin interactions and smooth muscle cells contraction by maintaining the activity of myosin light-chain kinase, independently of the free cytosolic calcium level. The sensitization of smooth muscle myofilaments to calcium has been implicated in many pathological states, such as hypertension, diabetes, heart attack, stroke, pulmonary hypertension, erectile dysfunction, and cancer. The focus of this review is on the involvement of RhoA/Rho-kinase in diseases. We will briefly describe the ROCK isoforms and the role of RhoA/Rho-kinase in the vasculature, before exploring the most recent findings regarding this pathway and various diseases.

Hypertension and Erectile Dysfunction: Breaking Down the Challenges.

A diagnostic of hypertension increases the risk of erectile dysfunction (ED); likewise, ED can be an early sign of hypertension. In both cases, there is evidence that endothelial dysfunction is a common link between the 2 conditions. During hypertension, the sustained and widespread release of procontractile factors (e.g., angiotensin II, endothelin 1, and aldosterone) impairs the balance between vasoconstrictors and vasodilators and, in turn, detrimentally impacts vascular and erectile structures. This prohypertensive state associates with an enhancement in the generation of reactive oxygen species, which is not compensated by internal antioxidant mechanisms. Recently, the innate immune system, mainly via Toll-like receptor 4, has also been shown to actively contribute to the pathophysiology of hypertension and ED not only by inducing oxidative stress but also by sustaining a low-grade inflammatory state. Furthermore, some drugs used to treat hypertension can cause ED and, consequently, reduce compliance with the prescribed pharmacotherapy. To break down these challenges, in this review, we focus on discussing the well-established as well as the emerging mechanisms linking hypertension and ED with an emphasis on the signaling network of the vasculature and corpora cavernosa, the vascular-like structure of the penis.

Crosstalk of TLR4, vascular NADPH oxidase, and COVID-19 in diabetes: What are the potential implications?

Toll-like receptor 4 (TLR4) contributes to the pathophysiology of diabetes. This happens, at least in part, because TLR4 modulates the enzyme NADPH oxidase, a primary source of ROS in vascular structures. Increased oxidative stress disrupts key vascular signaling mechanisms and drives the progression of diabetes, elevating the likelihood of cardiovascular diseases. Recently, it has been shown that patients with diabetes are also at a higher risk of developing severe coronavirus disease 2019 (COVID-19). Given the importance of the interaction between TLR4 and NADPH oxidase to the disrupted diabetic vascular system, we put forward the hypothesis that TLR4-mediated NADPH oxidase-derived ROS might be a critical mechanism to help explain why this disparity appears in diabetic patients, but unfortunately, conclusive experimental evidence still lacks in the literature. Herein, we focus on discussing the pathological implications of this signaling communication in the diabetic vasculature and exploring this crosstalk in the context of diabetes-associated severe COVID-19.

New insights into RhoA/Rho-kinase signaling: a key regulator of vascular contraction.

While Rho-signalling controlling vascular contraction is a canonical mechanism, with the modern approaches used in research, we are advancing our understanding and details into this pathway are often uncovered. RhoA-mediated Rho-kinase is the major regulator of vascular smooth muscle cells and a key player manoeuvring other functions in these cells. The discovery of new interactions, such as oxidative stress and hydrogen sulphide with Rho signalling are emerging addition not only in the physiology of the smooth muscle, but especially in the pathophysiology of vascular diseases. Likewise, the interplay between ageing and Rho-kinase in the vasculature has been recently considered. Importantly, in smooth muscle contraction, this pathway may also be affected by sex hormones, and consequently, sex-differences. This review provides an overview of Rho signalling mediating vascular contraction and focuses on recent topics discussed in the literature affecting this pathway such as ageing, sex differences and oxidative stress.

Impaired HSP70 Expression in the Aorta of Female Rats: A Novel Insight Into Sex-Specific Differences in Vascular Function.

Heat-shock protein 70 (HSP70) contributes to cellular calcium (Ca2+) handling mechanisms during receptor-mediated vascular contraction. Interestingly, previous studies have independently reported sex-related differences in HSP70 expression and Ca2+ dynamics. Still, it is unknown if sex, as a variable, plays a role in the impact that HSP70 has upon vascular contraction. To narrow this gap, we investigated if differences exist in the expression levels of HSP70 in the aorta, and if targeting this protein contributes to sex disparity in vascular responses. We report that, compared with male animals, female rats present a reduction in the basal levels of HSP70. More compelling, we found that the blockade of HSP70 has a greater impact on phenylephrine-induced phasic and tonic vascular contraction in female animals. In fact, it seems that the inhibition of HSP70 significantly affects vascular Ca2+ handling mechanisms in females, which could be associated with the fact that these animals have impaired HSP70 expression. Corroborating this idea, we uncovered that the higher sensitivity of female rats to HSP70 inhibition does not involve an increase in NO-dependent vasodilation nor a decrease in vascular oxidative stress. In summary, our findings reveal a novel mechanism associated with sex-specific differences in vascular responses to α-1 adrenergic stimulation, which might contribute to unraveling the network of intertwined pathways conferring female protection to (cardio)vascular diseases.

An additional physiological role for HSP70: Assistance of vascular reactivity.

AIMS: HSP70, a molecular chaperone, helps to maintain proteostasis. In muscle biology, however, evidence suggests HSP70 to have a more versatile range of functions, as genetic deletion of its inducible genes impairs Ca2+ handling, and consequently, cardiac and skeletal muscle contractility. Still, it is unknown whether HSP70 is involved in vascular reactivity, an intrinsic physiological mechanism of blood vessels. Therefore, we designed this study to test the hypothesis that proper vascular reactivity requires the assistance of HSP70. MAIN METHODS: We performed functional studies in a wire-myograph using thoracic aorta isolated from male Sprague Dawley rats. Experiments were conducted with and without an HSP70 inhibitor as well as in heat-stressed vessels. The expression levels of HSP70 were evaluated with Western blotting. NO and ROS levels were assessed with fluorescence microscopy. KEY FINDINGS: We report that blockade of HSP70 weakens contraction in response to phenylephrine (dose-response) in the aorta. Additionally, we demonstrated that inhibition of HSP70 affects the amplitude of the fast and of the slow components of the time-force curve. Corroborating these findings, we found that inhibition of HSP70, in vessels over-expressing this protein, partly rescues the contractile phenotype of aortic rings. Furthermore, we show that blockade of HSP70 facilitates relaxation in response to acetylcholine and clonidine without affecting the basal levels of NO and ROS. SIGNIFICANCE: Our work introduces an additional physiological role for HSP70, the assistance of vascular reactivity, which highlights this protein as a new player in vascular physiology, and therefore, uncovers a promising research avenue for vascular diseases.

Blockade of the TLR4-MD2 complex lowers blood pressure and improves vascular function in a murine model of type 1 diabetes.

While the pathogenesis of diabetes-induced high blood pressure (BP) is not entirely clear, current evidence suggests that Toll-like receptor 4 (TLR4) is a key player in the mechanisms associated with hypertension. However, it is unknown whether this receptor affects BP under type 1 diabetes. Likewise, there is insufficient knowledge about the role of TLR4 in diabetes-associated vascular dysfunction of large arteries. To narrow these gaps, in this study, we investigated if blockade of the TLR4-MD2 complex impacts BP and vascular function in diabetic rats. We injected streptozotocin in male Sprague Dawley rats and treated them with a neutralizing anti-TLR4 antibody for 14 days. BP was directly measured in conscious animals at the end of the treatment. In another set of experiments, we excised the aorta from control and diabetic animals, and measured TLR4 and MD2-a co-receptor that confers functionality to TLR4-levels by Western blotting. We also performed functional studies and evaluated ROS levels with and without a pharmacological inhibitor for TLR4 as well as for MD2. Additionally, we scrutinized a large human RNA-Seq dataset of aortic tissue to assess the co-expression of TLR4, MD2, and subunits of the vascular NADPH oxidases under diabetes and hypertension. We report that (a) chronic blockade of the TLR4-MD2 complex lowers BP in diabetic animals; that (b) type 1 diabetes modulates the levels of MD2 expression in the aorta, but not TLR4, at least in the conditions evaluated in this study; and, that (c) acute inhibition of TLR4 or MD2 diminishes vascular contractility and reduces oxidative stress in the aorta of these animals. In summary, we show evidence that the TLR4-MD2 complex is involved in the mechanisms linking type 1 diabetes and hypertension.

Blockade of Toll-like receptor 4 (TLR4) reduces oxidative stress and restores phospho-ERK1/2 levels in Leydig cells exposed to high glucose.

AIMS: Hyperglycemia in combination with oxidative stress plays a significant pathophysiological role in diabetic testicular dysfunction, often leading to infertility. Activation of Toll-like receptor 4 (TLR4) has been reported to mediate oxidative stress during diabetes. However, engagement of the TLR4 signaling pathway in diabetic testicular dysfunction has not been previously explored. Herein, we investigated the role of TLR4 in reactive oxygen species (ROS) production and in the phosphorylation status of ERK1/2 in primary Leydig cells exposed to high glucose and in testis isolated from diabetic rats. MAIN METHODS: Testicular levels of TLR4 and phospho-ERK1/2 were determined by Western blotting. ROS production was detected with a fluorescent probe. Additionally, primary Leydig cells were exposed to normal (5.5 mmol/l) or elevated (33 mmol/l) glucose concentrations and treated with or without a TLR4 inhibitor, CLI095 (10-5 mol/l) for 24 h, followed by evaluation of TLR4 and phospho-ERK1/2 expression levels by Western blotting and immunofluorescence staining, respectively. KEY FINDINGS: We show that high glucose induces the expression of TLR4 in Leydig cells. Additionally, we demonstrate that blockade of this receptor in this cell population reduces oxidative stress and restores the levels of phospho-ERK1/2. SIGNIFICANCE: Our findings provide new insight into TLR4 interaction with ROS and MEK/ERK pathway in Leydig cells exposed to high glucose and present a rationale for the development of new therapeutics for diabetic testicular dysfunction.

Angiotensin (1-7) Inhibits Ang II-mediated ERK1/2 Activation by Stimulating MKP-1 Activation in Vascular Smooth Muscle Cells.

The renin-angiotensin system (RAS) exerts profound physiological effects on blood pressure regulation and fluid homeostasis, mainly by modulating renal, cardiovascular, and central nervous systems. Angiotensin (Ang)-(1-7), an end-product of RAS, is recognized by its cardiovascular protective properties through stimulation of the Mas receptor, including vasodilation, anti-inflammatory, and antihypertensive actions, and consequently, counter-regulating the well-known Ang II-elicited actions. The overall hypothesis of this study is that Ang-(1-7) inhibits Ang II-induced ERK1/2 activation in vascular smooth muscle cells (VSMCs), via regulation of mitogen-activated protein phosphatase-1 (MKP-1) activity. Aortas from male Wistar rats were incubated with Ang-(1-7) or vehicle. Concentration-response curves to Ang II were performed in endothelium-denuded aortas, in the presence or absence of ERK1/2 (PD98059) inhibitor or Mas receptor (A-779) antagonist. Expression of proteins was assessed by western blot, and immunohistochemistry was conducted in VSMCs. Ang-(1-7) incubation decreased Ang II-induced contractile response in aortas, and this effect was not observed in the presence of PD98059 or A-779. Stimulation of VSMCs with Ang-(1-7) prevented Ang II-induced ERK1/2 phosphorylation, but not C-Raf-activation. Furthermore, Ang II decreased MKP-1 phosphorylation in VSMCs. Interestingly, simultaneous incubation of Ang-(1-7) with Ang II favored MKP-1 phosphorylation, negatively modulating ERK1/2 activation in VSMCs. The results suggest that Ang-(1-7) counter-regulates actions evoked by Ang II overproduction, as observed in cardiovascular diseases, mainly by modulating MKP-1 activity. This evidence suggests that the role of Ang-(1-7) in MKP-1-regulation represents a target for new therapeutic development.