Defective protein kinase C (PKC) signaling has been suggested to contribute to abnormal vascular contraction in disease conditions including hypertension and diabetes. Our previous work on agonist ...and pressure-induced cerebral vasoconstriction implicated PKC as a major contributor to force production in a myosin light chain (LC20) phosphorylation-independent manner. Here, we used phorbol dibutyrate to selectively induce a PKC-dependent constriction in rat middle cerebral arteries and delineate the relative contribution of different contractile mechanisms involved. Specifically, we employed an ultra-sensitive 3-step western blotting approach to detect changes in the content of phosphoproteins that regulate myosin light chain phosphatase (MLCP) activity, thin filament activation, and actin cytoskeleton reorganization. Data indicate that PKC activation evoked a greater constriction at a similar level of LC20 phosphorylation achieved by 5-HT. PDBu-evoked constriction persisted in the presence of Gö6976, a selective inhibitor of Ca2+-dependent PKC, and in the absence of extracellular Ca2+. Biochemical evidence indicates that either + or − extracellular Ca2+, PDBu (i) inhibits MLCP activity via the phosphorylation of myosin targeting subunit of myosin phosphatase (MYPT1) and C-kinase potentiated protein phosphatase-1 inhibitor (CPI-17), (ii) increases the phosphorylation of paxillin and heat shock protein 27 (HSP27), and reduces G-actin content, and (iii) does not change the phospho-content of the thin filament proteins, calponin and caldesmon. PDBu-induced constriction was more sensitive to disruption of actin cytoskeleton compared to inhibition of cross-bridge cycling. In conclusion, this study provided evidence for the pivotal contribution of cytoskeletal actin polymerization in force generation following PKC activation in cerebral resistance arteries.
Cardiac autonomic neuropathy (CAN) is an early cardiovascular manifestation of type 2 diabetes (T2D) that constitutes an independent risk factor for cardiovascular mortality and morbidity. ...Nevertheless, its underlying pathophysiology remains poorly understood. We recently showed that localized perivascular adipose tissue (PVAT) inflammation underlies the incidence of parasympathetic CAN in prediabetes. Here, we extend our investigation to provide a mechanistic framework for the evolution of autonomic impairment as the metabolic insult worsens. Early metabolic dysfunction was induced in rats fed a mild hypercaloric diet. Two low-dose streptozotocin injections were used to evoke a state of late decompensated T2D. Cardiac autonomic function was assessed by invasive measurement of baroreflex sensitivity using the vasoactive method. Progression into T2D was associated with aggravation of CAN to include both sympathetic and parasympathetic arms. Unlike prediabetic rats, T2D rats showed markers of brainstem neuronal injury and inflammation as well as increased serum levels of IL-1β. Experiments on PC12 cells differentiated into sympathetic-like neurons demonstrated that brainstem injury observed in T2D rats resulted from exposure to possible proinflammatory mediators in rat serum rather than a direct effect of the altered metabolic profile. CAN and the associated cardiovascular damage in T2D only responded to combined treatment with insulin to manage hyperglycemia in addition to a nonhypoglycemic dose of metformin or pioglitazone providing an anti-inflammatory effect, coincident with the effect of these combinations on serum IL-1β. Our present results indicate that CAN worsening upon progression to T2D involves brainstem inflammatory changes likely triggered by systemic inflammation.
Traumatic brain injury (TBI) is a major cause of disability and death. Mild TBI (mTBI) constitutes ~75% of all TBI cases. Repeated exposure to mTBI (rmTBI), leads to the exacerbation of the symptoms ...compared to single mTBI. To date, there is no FDA-approved drug for TBI or rmTBI. This research aims to investigate possible rmTBI neurotherapy by targeting TBI pathology-related mechanisms. Oxidative stress is partly responsible for TBI/rmTBI neuropathologic outcomes. Thus, targeting oxidative stress may ameliorate TBI/rmTBI consequences. In this study, we hypothesized that mitoquinone (MitoQ), a mitochondria-targeted antioxidant, would ameliorate TBI/rmTBI associated pathologic features by mitigating rmTBI-induced oxidative stress. To model rmTBI, C57BL/6 mice were subjected to three concussive head injuries. MitoQ (5 mg/kg) was administered intraperitoneally to rmTBI+MitoQ mice twice per week over one month. Behavioral and cognitive outcomes were assessed, 30 days following the first head injury, using a battery of behavioral tests. Immunofluorescence was used to assess neuroinflammation and neuronal integrity. Also, qRT-PCR was used to evaluate the expression levels of antioxidant enzymes. Our findings indicated that MitoQ alleviated fine motor function and learning impairments caused by rmTBI. Mechanistically, MitoQ reduced astrocytosis, microgliosis, dendritic and axonal shearing, and increased the expression of antioxidant enzymes. MitoQ administration following rmTBI may represent an efficient approach to ameliorate rmTBI neurological and cellular outcomes with no observable side effects.
•MitoQ alleviated fine motor function and learning impairments caused by rmTBI•MitoQ reduced neuroinflammation nen neurite shearing, and increased the expression of antioxidant enzymes•MitoQ may be tefficient to ameliorate rmTBIconsequences•MitoQ may pose its effect byaffecting mitochondrial complexes
The increased global prevalence of metabolic disorders including obesity, insulin resistance, metabolic syndrome and diabetes is mirrored by an increased incidence of prostate cancer (PCa). Ample ...evidence suggests that these metabolic disorders, being characterized by adipose tissue (AT) expansion and inflammation, not only present as risk factors for the development of PCa, but also drive its increased aggressiveness, enhanced progression, and metastasis. Despite the emerging molecular mechanisms linking AT dysfunction to the various hallmarks of PCa, thromboinflammatory processes implicated in the crosstalk between these diseases have not been thoroughly investigated. This is of particular importance as both diseases present states of hypercoagulability. Accumulating evidence implicates tissue factor, thrombin, and active factor X as well as other players of the coagulation cascade in the pathophysiological processes driving cancer development and progression. In this regard, it becomes pivotal to elucidate the thromboinflammatory processes occurring in the periprostatic adipose tissue (PPAT), a fundamental microenvironmental niche of the prostate. Here, we highlight key findings linking thromboinflammation and the pleiotropic effects of coagulation factors and their inhibitors in metabolic diseases, PCa, and their crosstalk. We also propose several novel therapeutic targets and therapeutic interventions possibly modulating the interaction between these pathological states.
Display omitted
Hypertension is a major risk factor for cardiovascular disease (CVD) as well as a major contributor to all-cause mortality and disability worldwide. The pathophysiology of ...hypertension is highly attributed to a dysfunctional endothelium and vascular remodeling. Despite the wide use of pharmacological therapies that modulate these pathways, a large percentage of patients continue to have uncontrolled hypertension, and the use of non-pharmacological interventions is increasingly investigated. Among these, caloric restriction (CR) appears to be a promising nutritional intervention for the management of hypertension. However, the mechanisms behind this effect are not yet fully understood, although an evolving view supports a significant impact of CR on vascular structure and function, specifically at the level of vascular endothelial cells, vascular smooth muscle cells along with their extracellular matrix (ECM). Accumulating evidence suggests that CR promotes endothelium-dependent vasodilation through activating eNOS and increasing nitric oxide (NO) levels through multiple cascades involving modulation of oxidative stress, autophagy, and inflammation. Indeed, CR diminishes phenotypic shift, and suppresses proliferation and migration of VSMCs via pathways involving NO and mTOR. By regulating transforming growth factor-β and matrix metalloproteinases, CR appears to reduce ECM and collagen deposition in vascular walls. Here, we offer a detailed discussion of how these mechanisms contribute to CR’s influence on reducing blood pressure. Such mechanisms could then provide a valuable foundation on which to base new therapeutic interventions for hypertension.
The use of complementary medicine (CMD) for liver support in Hepatitis C virus (HCV) patients sometimes coincides with the administration of oral antiviral drugs to eradicate the virus. This calls ...for a deep investigation of CMD effects on the pharmacokinetic parameters of these drugs to ensure their safety and efficacy. Silymarin (SLY), as a CMD, was selected to be given orally to healthy male rats with sofosbuvir (SFB) and ledipasvir (LED), a common regimen in HCV treatment. A new and sensitive LC–MS method was validated for the bioassay of SLY, LED, SFB and its inactive metabolite, GS‐331007, in spiked plasma with lower limits of quantitation of 10, 1, 4 and 10 ng/ml, respectively. Moreover, the method was further applied to conduct a full pharmacokinetic profile of SFB, GS‐331007 and ledipasvir with and without SLY. It was found that co‐administration of SLY may expose the patient to unplanned high serum concentrations of SFB and LED. This could be accompanied by a decrease in SFB efficacy, potentially leading to therapeutic failure and the emergence of viral resistance.
Traumatic brain injury (TBI) is a global health burden and a major cause of disability and mortality. An early cascade of physical and structural damaging events starts immediately post-TBI. This ...primary injury event initiates a series of neuropathological molecular and biochemical secondary injury sequelae, that last much longer and involve disruption of cerebral metabolism, mitochondrial dysfunction, oxidative stress, neuroinflammation, and can lead to neuronal damage and death. Coupled to these events, recent studies have shown that lifestyle factors, including diet, constitute additional risk affecting TBI consequences and neuropathophysiological outcomes. There exists molecular cross-talk among the pathways involved in neuronal survival, neuroinflammation, and behavioral outcomes, that are shared among western diet (WD) intake and TBI pathophysiology. As such, poor dietary intake would be expected to exacerbate the secondary damage in TBI. Hence, the aim of this review is to discuss the pathophysiological consequences of WD that can lead to the exacerbation of TBI outcomes. We dissect the role of mitochondrial dysfunction, oxidative stress, neuroinflammation, and neuronal injury in this context. We show that currently available data conclude that intake of a diet saturated in fats, pre- or post-TBI, aggravates TBI, precludes recovery from brain trauma, and reduces the response to treatment.
Our understanding of the cellular signalling mechanisms contributing to agonist‐induced constriction is almost exclusively based on the study of conduit arteries. Resistance arteries/arterioles have ...received less attention as standard biochemical approaches lack the necessary sensitivity to permit quantification of phosphoprotein levels in these small vessels. Here, we have employed a novel, highly sensitive Western blotting method to assess: (1) the contribution of Ca2+ sensitization mediated by phosphorylation of myosin light chain phosphatase targeting subunit 1 (MYPT1) and the 17 kDa PKC‐potentiated protein phosphatase 1 inhibitor protein (CPI‐17) to serotonin (5‐HT)‐induced constriction of rat middle cerebral arteries, and (2) whether there is any interplay between pressure‐induced myogenic and agonist‐induced mechanisms of vasoconstriction. Arterial diameter and levels of MYPT1 (T697 and T855), CPI‐17 and 20 kDa myosin light chain subunit (LC20) phosphorylation were determined following treatment with 5‐HT (1 μmol l−1) at 10 or 60 mmHg in the absence and presence of H1152 or GF109203X to suppress the activity of Rho‐associated kinase (ROK) and protein kinase C (PKC), respectively. Although H1152 and GF109203X suppressed 5‐HT‐induced constriction and reduced phospho‐LC20 content at 10 mmHg, we failed to detect any increase in MYPT1 or CPI‐17 phosphorylation. In contrast, an increase in MYPT1‐T697 and MYPT1‐T855 phosphorylation, but not phospho‐CPI‐17 content, was apparent at 60 mmHg following exposure to 5‐HT, and the phosphorylation of both MYPT1 sites was sensitive to H1152 inhibition of ROK. The involvement of MYPT1 phosphorylation in the response to 5‐HT at 60 mmHg was not dependent on force generation per se, as inhibition of cross‐bridge cycling with blebbistatin (10 μmol l−1) did not affect phosphoprotein content. Taken together, the data indicate that Ca2+ sensitization owing to ROK‐mediated phosphorylation of MYPT1 contributes to 5‐HT‐evoked vasoconstriction only in the presence of pressure‐induced myogenic activation. These findings provide novel evidence of an interplay between myogenic‐ and agonist‐induced vasoconstriction in cerebral resistance arteries.
Blood pressure and organ‐specific blood flow regulation are mediated by precise changes in the diameter of small arterial vessels. Vessel diameter is a function of the level of vascular smooth muscle contraction due to intrinsic pressure‐dependent (myogenic) mechanisms and in response to extrinsic vasoactive substances. We show that there is an interplay between vasoactive serotonin‐ and pressure‐induced constriction at the level of Rho kinase‐mediated inhibition of myosin light chain phosphatase leading to calcium sensitization of the contractile apparatus and increased force generation. Knowledge of the molecular mechanisms involved in the integration of divergent vasoregulatory signals increases our understanding of how blood pressure and blood flow are regulated and will help us to identify the basis of dysfunctional control in disease.
Endothelial dysfunction is a hallmark of diabetic vasculopathies. Although hyperglycemia is believed to be the culprit causing endothelial damage, the mechanism underlying early endothelial insult in ...prediabetes remains obscure. We used a nonobese high-calorie (HC)-fed rat model with hyperinsulinemia, hypercholesterolemia, and delayed development of hyperglycemia to unravel this mechanism. Compared with aortic rings from control rats, HC-fed rat rings displayed attenuated acetylcholine-mediated relaxation. While sensitive to nitric oxide synthase (NOS) inhibition, aortic relaxation in HC-rat tissues was not affected by blocking the inward-rectifier potassium (Kir) channels using BaCl
Although Kir channel expression was reduced in HC-rat aorta, Kir expression, endothelium-dependent relaxation, and the BaCl
-sensitive component improved in HC rats treated with atorvastatin to reduce serum cholesterol. Remarkably, HC tissues demonstrated increased reactive species (ROS) in smooth muscle cells, which was reversed in rats receiving atorvastatin. In vitro ROS reduction, with superoxide dismutase, improved endothelium-dependent relaxation in HC-rat tissues. Significantly, connexin-43 expression increased in HC aortic tissues, possibly allowing ROS movement into the endothelium and reduction of eNOS activity. In this context, gap junction blockade with 18-
-glycyrrhetinic acid reduced vascular tone in HC rat tissues but not in controls. This reduction was sensitive to NOS inhibition and SOD treatment, possibly as an outcome of reduced ROS influence, and emerged in BaCl
-treated control tissues. In conclusion, our results suggest that early metabolic challenge leads to reduced Kir-mediated endothelium-dependent hyperpolarization, increased vascular ROS potentially impairing NO synthesis and highlight these channels as a possible target for early intervention with vascular dysfunction in metabolic disease. SIGNIFICANCE STATEMENT: The present study examines early endothelial dysfunction in metabolic disease. Our results suggest that reduced inward-rectifier potassium channel function underlies a defective endothelium-mediated relaxation possibly through alteration of nitric oxide synthase activity. This study provides a possible mechanism for the augmentation of relatively small changes in one endothelium-mediated relaxation pathway to affect overall endothelial response and highlights the potential role of inward-rectifier potassium channel function as a therapeutic target to treat vascular dysfunction early in the course of metabolic disease.
PDF
