pH-sensing GPR68 inhibits vascular smooth muscle cell proliferation through Rap1A.

Phenotypic transformation of vascular smooth muscle (VSM) from a contractile state to a synthetic, proliferative state is a hallmark of cardiovascular disease (CVD). In CVD, diseased tissue often becomes acidic from altered cellular metabolism secondary to compromised blood flow, yet the contribution of local acid/base imbalance to the disease process has been historically overlooked. In this study, we examined the regulatory impact of the pH-sensing G protein-coupled receptor GPR68 on vascular smooth muscle (VSM) proliferation in vivo and in vitro in wild-type (WT) and GPR68 knockout (KO) male and female mice. Arterial injury reduced GPR68 expression in WT vessels and exaggerated medial wall remodeling in GPR68 KO vessels. In vitro, KO VSM cells showed increased cell cycle progression and proliferation compared to WT VSM cells, and GPR68-inducing acidic exposure reduced proliferation in WT cells. mRNA and protein expression analyses revealed increased Rap1A in KO cells compared to WT cells, and RNA silencing of Rap1A reduced KO VSM cell proliferation. In sum, these findings support a growth-inhibitory capacity of pH-sensing GPR68 and suggest a mechanistic role for the small GTPase Rap1A in GPR68-mediated VSM growth control. These results shed light on GPR68 and its effector Rap1A as potential targets to combat pathologic phenotypic switching and proliferation in VSM.

Emerging Role of Kinin B1 Receptor in Persistent Neuroinflammation and Neuropsychiatric Symptoms in Mice Following Recovery from SARS-CoV-2 Infection.

Evidence suggests that patients with long COVID can experience neuropsychiatric, neurologic, and cognitive symptoms. However, these clinical data are mostly associational studies complicated by confounding variables, thus the mechanisms responsible for persistent symptoms are unknown. Here we establish an animal model of long-lasting effects on the brain by eliciting mild disease in K18-hACE2 mice. Male and female K18-hACE2 mice were infected with 4 × 103 TCID50 of SARS-CoV-2 and, following recovery from acute infection, were tested in the open field, zero maze, and Y maze, starting 30 days post infection. Following recovery from SARS-CoV-2 infection, K18-hACE2 mice showed the characteristic lung fibrosis associated with SARS-CoV-2 infection, which correlates with increased expression of the pro-inflammatory kinin B1 receptor (B1R). These mice also had elevated expression of B1R and inflammatory markers in the brain and exhibited behavioral alterations such as elevated anxiety and attenuated exploratory behavior. Our data demonstrate that K18-hACE2 mice exhibit persistent effects of SARS-CoV-2 infection on brain tissue, revealing the potential for using this model of high sensitivity to SARS-CoV-2 to investigate mechanisms contributing to long COVID symptoms in at-risk populations. These results further suggest that elevated B1R expression may drive the long-lasting inflammatory response associated with SARS-CoV-2 infection.

Cardiomyocyte-specific deletion of TLR4 attenuates angiotensin II-induced hypertension and cardiac remodeling.

Toll-like receptor 4 (TLR4) is an integral factor in the initiation of the innate immune response and plays an important role in cardiovascular diseases such as hypertension and myocardial infarction. Previous studies from our lab demonstrated that central TLR4 blockade reduced cardiac TLR4 expression, attenuated hypertension, and improved cardiac function. However, the contribution of cardiac specific TLR4 to the development of hypertension and cardiac remodeling is unknown. Therefore, we hypothesized that cardiomyocyte specific knockdown of TLR4 would have beneficial effects on hypertension, cardiac hypertrophy, and remodeling. To test this hypothesis, cardiomyocyte-specific TLR4 knockdown (cTLR4KO) mice were generated by crossing floxed TLR4 mice with Myh6-Cre mice, and subjected to angiotensin II (Ang II, 1 µg/kg/min or vehicle for 14 days) hypertension model. Blood pressure measurements using radio telemetry revealed no differences in baseline mean arterial pressure between control littermates and cTLR4KO mice (103 ± 2 vs. 105 ± 3 mmHg, p > 0.05). Ang II-induced hypertension (132 ± 2 vs. 151 ± 3 mmHg, p < 0.01) was attenuated and cardiac hypertrophy (heart/body weight; 4.7 vs. 5.8 mg/g, p < 0.01) was prevented in cTLR4KO mice when compared with control mice. In addition, the level of myocardial fibrosis was significantly reduced, and the cardiac function was improved in cTLR4KO mice infused with Ang II. Furthermore, cardiac inflammation, as evidenced by elevated gene expression of TNF, IL-6, and MCP-1 in the left ventricle, was attenuated in cTLR4KO mice infused with Ang II. Together, this data revealed a protective role for cardiomyocyte-specific deletion of TLR4 against Ang II-induced hypertension and cardiac dysfunction through inhibition of proinflammatory cytokines.

Kinin B1 Receptor Mediates Bidirectional Interaction between Neuroinflammation and Oxidative Stress.

Hypertension is associated with increased expression of kinin B1 receptors (B1R) and increased levels of pro-inflammatory cytokines within the neurons. We previously reported that angiotensin II (Ang II) upregulates B1R expression and can induce neuroinflammation and oxidative stress in primary hypothalamic neurons. However, the order in which B1R activation, neuroinflammation, and oxidative stress occur has not yet been studied. Using primary hypothalamic neurons from neonatal mice, we show that tumor necrosis factor (TNF), lipopolysaccharides (LPS), and hydrogen peroxide (H2O2) can upregulate B1R expression and increase oxidative stress. Furthermore, our study shows that B1R blockade with R715, a specific B1R antagonist, can attenuate these effects. To further confirm our findings, we used a deoxycorticosterone acetate (DOCA)-salt model of hypertension to show that oxidative stress is upregulated in the hypothalamic paraventricular nucleus (PVN) of the brain. Together, these data provide novel evidence that relationship between oxidative stress, neuroinflammation, and B1R upregulation in the brain is bidirectional, and that B1R antagonism may have beneficial effects on neuroinflammation and oxidative stress in various disease pathologies.

Kinin B1R Activation Induces Endoplasmic Reticulum Stress in Primary Hypothalamic Neurons.

The endoplasmic reticulum (ER) is a key organelle involved in homeostatic functions including protein synthesis and transport, and the storage of free calcium. ER stress potentiates neuroinflammation and neurodegeneration and is a key contributor to the pathogenesis of neurogenic hypertension. Recently, we showed that kinin B1 receptor (B1R) activation plays a vital role in modulating neuroinflammation and hypertension. However, whether B1R activation results in the progression and enhancement of ER stress has not yet been studied. In this brief research report, we tested the hypothesis that B1R activation in neurons contributes to unfolded protein response (UPR) and the development of ER stress. To test this hypothesis, we treated primary hypothalamic neuronal cultures with B1R specific agonist Lys-Des-Arg9-Bradykinin (LDABK) and measured the components of UPR and ER stress. Our data show that B1R stimulation via LDABK, induced the upregulation of GRP78, a molecular chaperone of ER stress. B1R stimulation was associated with an increased expression and activation of transmembrane ER stress sensors, ATF6, IRE1α, and PERK, the critical components of UPR. In the presence of overwhelming ER stress, activated ER stress sensors can lead to oxidative stress, autophagy, or apoptosis. To determine whether B1R activation induces apoptosis we measured intracellular Ca2+ and extracellular ATP levels, caspases 3/7 activity, and cell viability. Our data show that LDABK treatment does increase Ca2+ and ATP levels but does not alter caspase activity or cell viability. These findings suggest that B1R activation initiates the UPR and is a key factor in the ER stress pathway.