RESUMEN
Heat-shock proteins (HSPs) are primary stress responders that are vital to maintaining homeostasis [...].
Asunto(s)
Proteínas HSP70 de Choque Térmico , Proteínas de Choque Térmico , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Transducción de Señal , Proteínas HSP90 de Choque Térmico/metabolismoRESUMEN
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.
Asunto(s)
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Animales , Ratas , Calcio , Proteínas HSP70 de Choque Térmico/metabolismo , Estreptozocina , Receptor Toll-Like 4/metabolismoRESUMEN
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 = â¼ 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.
RESUMEN
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.
Asunto(s)
Diabetes Mellitus , Hiperglucemia , Resistencia a la Insulina , Diabetes Mellitus/tratamiento farmacológico , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Transducción de Señal/fisiologíaRESUMEN
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.
Asunto(s)
Vasos Sanguíneos/enzimología , COVID-19/virología , Diabetes Mellitus/enzimología , Angiopatías Diabéticas/enzimología , NADPH Oxidasas/metabolismo , SARS-CoV-2/patogenicidad , Receptor Toll-Like 4/metabolismo , Animales , Vasos Sanguíneos/fisiopatología , Vasos Sanguíneos/virología , COVID-19/enzimología , COVID-19/fisiopatología , Diabetes Mellitus/fisiopatología , Angiopatías Diabéticas/fisiopatología , Activación Enzimática , Interacciones Huésped-Patógeno , Humanos , Estrés Oxidativo , Pronóstico , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
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.
RESUMEN
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.
Asunto(s)
Contracción Muscular , Vasoconstricción , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Animales , HumanosRESUMEN
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.
Asunto(s)
Disfunción Eréctil , Hipertensión , Disfunción Eréctil/epidemiología , Humanos , Hipertensión/diagnóstico , Hipertensión/fisiopatología , MasculinoRESUMEN
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.
RESUMEN
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.
Asunto(s)
Presión Sanguínea , Vasos Sanguíneos/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/fisiopatología , Antígeno 96 de los Linfocitos/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 1/etiología , Modelos Animales de Enfermedad , Hipertensión/etiología , Hipertensión/metabolismo , Hipertensión/fisiopatología , Masculino , Ratones , Estrés Oxidativo , Unión Proteica , Ratas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
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.
Asunto(s)
Aorta Torácica/fisiología , Endotelio Vascular/fisiología , Proteínas HSP70 de Choque Térmico/fisiología , Músculo Liso Vascular/fisiología , Acetilcolina/farmacología , Animales , Aorta Torácica/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Endotelio Vascular/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/agonistas , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Masculino , Músculo Liso Vascular/efectos de los fármacos , Técnicas de Cultivo de Órganos , Fenilefrina/farmacología , Nucleósidos de Purina/farmacología , Ratas , Ratas Sprague-Dawley , Vasoconstrictores/farmacología , Vasodilatadores/farmacologíaRESUMEN
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.
Asunto(s)
Hiperglucemia/metabolismo , Células Intersticiales del Testículo/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Estrés Oxidativo , Receptor Toll-Like 4/antagonistas & inhibidores , Animales , Western Blotting , Diabetes Mellitus Experimental/metabolismo , Sistema de Señalización de MAP Quinasas , Masculino , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Receptor Toll-Like 4/metabolismoRESUMEN
Pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) and NOD-like receptors (NLRs) play crucial roles in the underlying mechanisms of metabolic syndrome (MetS). Mainly, these receptors have been suggested to participate in the pathophysiological processes involved in the complications associated with this condition. Therefore, to evolve therapeutic strategies targeting PRRs might be an imperative approach to avoid the development of further complications in human subjects. In this work, we discuss the understanding regarding the roles of PRRs in the pathways of MetS to further describe potential advancements made to target these receptors within this pathology.
Asunto(s)
Síndrome Metabólico/fisiopatología , Síndrome Metabólico/terapia , Receptores de Reconocimiento de Patrones/metabolismo , Animales , Humanos , Transducción de SeñalRESUMEN
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.
Asunto(s)
Sistema Cardiovascular/metabolismo , Biología Computacional , Proteínas HSP70 de Choque Térmico/metabolismo , Antígeno 96 de los Linfocitos/metabolismo , Modelos Biológicos , Receptor Toll-Like 4/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Biología Computacional/métodos , Femenino , Perfilación de la Expresión Génica , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/genética , Humanos , Antígeno 96 de los Linfocitos/química , Antígeno 96 de los Linfocitos/genética , Masculino , Persona de Mediana Edad , Modelos Moleculares , Complejos Multiproteicos/metabolismo , Unión Proteica , Relación Estructura-Actividad , Receptor Toll-Like 4/química , Receptor Toll-Like 4/genética , Transcriptoma , Adulto JovenRESUMEN
Hyperglycemia is a chief factor in diabetes, a complex disease associated with reproductive disorders, mainly testicular dysfunction, which contributes to male infertility. Leydig cells are the predominant cell population in the testis interstitium and, when stimulated, they are capable of initiating immune responses playing crucial roles in the mechanisms related to testis' homeostasis. These cells express TLR4, an innate immune receptor, which is known to be modulated by hyperglycemia in other cell populations and tissue types. Still, whether TLR4 contributes to hyperglycemia-associated testicular dysfunction remains elusive. Activation of TLR4 in response to high glucose levels involves redox imbalance and stimulation of transcriptional factors, especially nuclear factor (NF)-κB, which could be a potential pathological mechanism compromising the testis integrity. Additionally, emerging evidence shows crosstalk between TLR4 and Nrf2, an anti-inflammatory mediator that was previously shown to be reduced in diabetic testis. Therefore, we hypothesize that hyperglycemia-mediated TLR4 activation in testicular cells, especially Leydig cells might be a crucial event triggering oxidative stress and inflammation, which in turn, drives testicular dysfunction.
Asunto(s)
Hiperglucemia/metabolismo , Testículo/fisiopatología , Receptor Toll-Like 4/genética , Animales , Antioxidantes/metabolismo , Glucosa/metabolismo , Homeostasis , Humanos , Inmunidad Innata , Inflamación , Células Intersticiales del Testículo/metabolismo , Masculino , Ratones , FN-kappa B/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Receptor Toll-Like 4/metabolismoRESUMEN
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.
Asunto(s)
Diabetes Mellitus Experimental/tratamiento farmacológico , Disfunción Eréctil/tratamiento farmacológico , Péptidos/farmacología , Venenos de Araña/farmacología , Administración Intravenosa , Administración Tópica , Animales , GMP Cíclico/metabolismo , Disfunción Eréctil/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico/metabolismo , Erección Peniana/efectos de los fármacos , Inhibidores de Fosfodiesterasa 5/farmacología , Ratas , Ratas Endogámicas SHR , Ratas Sprague-Dawley , Ratas Wistar , Citrato de Sildenafil/farmacología , Estreptozocina , Distribución TisularRESUMEN
Diabetes is one of the most concerning diseases in modern times. Despite considerable advances in therapeutic management, the prevalence of diabetes and its contribution to death and disability continue to be a major health problem. Diabetic vasculopathies are the leading cause of mortality and morbidity in diabetic patients. Its pathophysiology includes oxidative stress, advanced glycation end products, and a low-grade inflammatory state. Lately, actions of the innate immune system via Toll-like receptors (TLRs) have been suggested as a new insight in this field. TLRs are pattern recognition receptors activated by highly conserved structural motifs of exogenous or endogenous ligands. Heat-shock proteins (HSPs), normally known for their ability to protect cells during stressful conditions, when released from injured cells bind to TLR4 and trigger the release of pro-inflammatory cytokines in a MyD88-dependent pathway. This pathway had been investigated in pancreatic beta cells and skeletal muscle, but it has not yet been explored in the vascular system and deserves investigation. In this work, the interplay between TLR4 and HSP70 in the vasculature during diabetes is reviewed and discussed. The current literature and preliminary results from our laboratory led us to hypothesize that hyperglycemia-associated HSP70 plays an important role in the pathophysiology of diabetic vasculopathies via the TLR4 pathway and might be a new target for therapeutic intervention.
Asunto(s)
Angiopatías Diabéticas/tratamiento farmacológico , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/antagonistas & inhibidores , Animales , Angiopatías Diabéticas/inmunología , Angiopatías Diabéticas/patología , Modelos Animales de Enfermedad , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/inmunología , Endotelio Vascular/patología , Productos Finales de Glicación Avanzada/inmunología , Productos Finales de Glicación Avanzada/metabolismo , Proteínas HSP70 de Choque Térmico/inmunología , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Terapia Molecular Dirigida/métodos , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/inmunología , Transducción de Señal/inmunología , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 4/metabolismoRESUMEN
The immune system plays a prominent role in the initiation and maintenance of hypertension. The innate immune system, via toll-like receptors (TLRs), identifies distinct signatures of invading microbes and damage-associated molecular patterns and triggers a chain of downstream signalling cascades, leading to secretion of pro-inflammatory cytokines and shaping the adaptive immune response. Over the past decade, a dysfunctional TLR-mediated response, particularly via TLR4, has been suggested to support a chronic inflammatory state in hypertension, inducing deleterious local and systemic effects in host cells and tissues and contributing to disease progression. While the underlying mechanisms triggering TLR4 need further research, evidence suggests that sustained elevations in BP disrupt homeostasis, releasing endogenous TLR4 ligands in hypertension. In this review, we discuss the emerging role of TLR4 in the pathogenesis of hypertension and whether targeting this receptor and its signalling pathways could offer a therapeutic strategy for management of this multifaceted disease. LINKED ARTICLES: This article is part of a themed section on Immune Targets in Hypertension. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.12/issuetoc.
Asunto(s)
Antihipertensivos/farmacología , Hipertensión/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/antagonistas & inhibidores , Animales , Humanos , Hipertensión/inmunología , Ligandos , Transducción de Señal/inmunología , Receptor Toll-Like 4/inmunologíaRESUMEN
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.