Search

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 with 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 with 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 pathological phenotypic switching and proliferation in VSM. NEW & NOTEWORTHY Extracellular acidosis remains an understudied feature of many pathologies. We examined a potential regulatory role for pH-sensing GPR68 in vascular smooth muscle (VSM) growth in the context of CVD. With in vivo and in vitro growth models with GPR68-deficient mice and GPR68 induction strategies, novel findings revealed capacity of GPR68 to attenuate growth through the small GTPase Rap1A. These observations highlight GPR68 and its effector Rap1A as possible therapeutic targets to combat pathological VSM growth.

Transition of arterial smooth muscle (ASM) from a quiescent, contractile state to a growth-promoting state is a hallmark of cardiovascular disease (CVD), a leading cause of death and disability in the United States and worldwide. While many individual signals have been identified as important mechanisms in this phenotypic conversion, the combined impact of the transcription factors Smad3 and FoxO3 in ASM growth is not known. The purpose of this study was to determine that a coordinated, phosphorylation-specific relationship exists between Smad3 and FoxO3 in the control of ASM cell growth. Using a rat in vivo arterial injury model and rat primary ASM cell lysates and fractions, validated low and high serum in vitro models of respective quiescent and growth states, and adenoviral (Ad-) gene delivery for overexpression (OE) of individual and combined Smad3 and/or FoxO3, we hypothesized that FoxO3 can moderate Smad3-induced ASM cell growth. Key findings revealed unique cellular distribution of Smad3 and FoxO3 under growth conditions, with induction of both nuclear and cytosolic Smad3 yet primarily cytosolic FoxO3; Ad-Smad3 OE leading to cytosolic and nuclear expression of phosphorylated and total Smad3, with almost complete reversal of each with Ad-FoxO3 co-infection in quiescent and growth conditions; Ad-FoxO3 OE leading to enhanced cytosolic expression of phosphorylated and total FoxO3, both reduced with Ad-Smad3 co-infection in quiescent and growth conditions; Ad-FoxO3 inducing expression and activity of the ubiquitin ligase MuRF-1, which was reversed with concomitant Ad-Smad3 OE; and combined Smad3/FoxO3 OE reversing both the pro-growth impact of singular Smad3 and the cytostatic impact of singular FoxO3. A primary takeaway from these observations is the capacity of FoxO3 to reverse growth-promoting effects of Smad3 in ASM cells. Additional findings lend support for reciprocal antagonism of Smad3 on FoxO3-induced cytostasis, and these effects are dependent upon discrete phosphorylation states and cellular localization and involve MuRF-1 in the control of ASM cell growth. Lastly, results showing capacity of FoxO3 to normalize Smad3-induced ASM cell growth largely support our hypothesis, and overall findings provide evidence for utility of Smad3 and/or FoxO3 as potential therapeutic targets against abnormal ASM growth in the context of CVD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Francisco, Holt, Bullock, Williams, Poovey, Holland, Brault and Tulis.)

Background: Living kidney donation (LKD) is a preferred treatment option for end-stage chronic kidney disease, but it can also pose potential risks for the donor, including hypertension and end-stage renal disease. Many donors are women of reproductive age who may have concerns about the effects of donation on future pregnancies. The aim of this study was to determine fetal and maternal outcomes in a cohort of pregnancies after LKD and to compare them with pregnancies before LKD., Methods: We conducted a retrospective analysis of living kidney donors of childbearing age (<46 years old) at the time of donation who got pregnant after LKD in our center between 1987 and 2020 (N = 13). Clinical data were collected, including demographic characteristics and maternal and fetal outcomes., Results: We observed 16 pregnancies after LKD and 12 pregnancies before LKD in the same group of patients. The rate of gestational hypertension was 12.5% in pregnancies after LKD and 8.3% before LKD (P = .999). There were 13 successful pregnancies after LKD with a mean gestational age of 38.6 ± 1.7 weeks. There were no episodes of acute kidney injury or other complications., Conclusion: The present study suggests that LKD does not have a negative effect on maternal and fetal outcomes. However, caution should be taken due to the small sample size. We agree with the guidelines recommending close monitoring of post-donation pregnancies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Stroke identification is a key step in acute ischemic stroke management. Our objectives were to prospectively examine the agreement between prehospital and hospital Modified National Institutes of Health Stroke Scale (mNIHSS) assessments as well as assess the prehospital performance characteristics of the mNIHSS for identification of large vessel occlusion strokes. Method: In this prospective cohort study conducted over a 20-month period (11/2016-6/2018), we trained 40 prehospital providers (paramedics) in Emergency Neurological Life Support (ENLS) curriculum and in mNIHSS. English-speaking patients aged 18 and above transported for an acute neurological deficit were included. Using unique identifiers, we linked the prehospital assessment records to the hospital record. We calculated the agreement between prehospital and hospital mNIHSS scores using the Bland-Altman analysis and the sensitivity and specificity of the prehospital mNIHSS. Results: Of the 31 patients, the mean difference (prehospital mNIHSS-hospital mNIHSS) was 2.4, 95% limits of agreement (-5.2 to 10.0); 10 patients (32%) met our a priori imaging definition of large vessel occlusion and the sensitivity of mNIHSS ≥ 8 was 6/10 or 0.60 (95% CI: 0.26-0.88) and the specificity was 13/21 or 0.62 (95% CI: 0.38-0.82), respectively. Conclusions: We were able to train prehospital providers to use the prehospital mNIHSS. Prehospital and hospital mNIHSS had a reasonable level of agreement and and the scale was able to predict large vessel occlusions with moderate sensitivity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mulkerin, Spokoyny, Francisco, Lima, Corry, Nudelman, Niknam, Brown, Kohn and Govindarajan.)

Cardiovascular disease (CVD), including myocardial infarction (MI) and peripheral or coronary artery disease (PAD, CAD), remains the number one killer of individuals in the United States and worldwide, accounting for nearly 18 million (>30%) global deaths annually. Despite considerable basic science and clinical investigation aimed at identifying key etiologic components of and potential therapeutic targets for CVD, the number of individuals afflicted with these dreaded diseases continues to rise. Of the many biochemical, molecular, and cellular elements and processes characterized to date that have potential to control foundational facets of CVD, the multifaceted cyclic nucleotide pathways continue to be of primary basic science and clinical interest. Cyclic adenosine monophosphate (cyclic AMP) and cyclic guanosine monophosphate (cyclic GMP) and their plethora of downstream protein kinase effectors serve ubiquitous roles not only in cardiovascular homeostasis but also in the pathogenesis of CVD. Already a major target for clinical pharmacotherapy for CVD as well as other pathologies, novel and potentially clinically appealing actions of cyclic nucleotides and their downstream targets are still being discovered. With this in mind, this review article focuses on our current state of knowledge of the cyclic nucleotide-driven serine (Ser)/threonine (Thr) protein kinases in CVD with particular emphasis on cyclic AMP-dependent protein kinase (PKA) and cyclic GMP-dependent protein kinase (PKG). Attention is given to the regulatory interactions of these kinases with inflammatory components including interleukin 6 signals, with G protein-coupled receptor and growth factor signals, and with growth and synthetic transcriptional platforms underlying CVD pathogenesis. This article concludes with a brief discussion of potential future directions and highlights the importance for continued basic science and clinical study of cyclic nucleotide-directed protein kinases as emerging and crucial controllers of cardiac and vascular disease pathologies., Competing Interests: The authors declare no conflicts of interest.

Application of fluid shear stress to adherent cells dramatically influences their cytoskeletal makeup and differentially regulates their migratory phenotype. Because cytoskeletal rearrangements are necessary for cell motility and migration, preserving these adaptations under in vitro conditions and in the presence of fluid flow are physiologically essential. With this in mind, parallel plate flow chambers and microchannels are often used to conduct in vitro perfusion experiments. However, both of these systems currently lack capacity to accurately study cell migration in the same location where cells were perfused. The most common perfusion/migration assays involve cell perfusion followed by trypsinization which can compromise adaptive cytoskeletal geometry and lead to misleading phenotypic conclusions. The purpose of this study was to quantitatively highlight some limitations commonly found with currently used cell migration approaches and to introduce two new advances which use additive manufacturing (3D printing) or laser capture microdissection (LCM) technology. The residue-free 3D printed insert allows accurate cell seeding within defined areas, increases cell yield for downstream analyses, and more closely resembles the reported levels of fluid shear stress calculated with computational fluid dynamics as compared to other residue-free cell seeding techniques. The LCM approach uses an ultraviolet laser for "touchless technology" to rapidly and accurately introduce a custom-sized wound area in otherwise inaccessible perfusion microchannels. The wound area introduced by LCM elicits comparable migration characteristics compared to traditional pipette tip-induced injuries. When used in perfusion experiments, both of these newly characterized tools were effective in yielding similar results yet without the limitations of the traditional modalities. These innovative methods provide valuable tools for exploring mechanisms of clinically important aspects of cell migration fundamental to the pathogenesis of many flow-mediated disorders and are applicable to other perfusion-based models where migration is of central importance. © 2016 International Society for Advancement of Cytometry., (© 2016 International Society for Advancement of Cytometry.)

Coronary artery disease (CAD) accounts for over half of all cardiovascular disease-related deaths. Uncontrolled arterial smooth muscle (ASM) cell migration is a major component of CAD pathogenesis and efforts aimed at attenuating its progression are clinically essential. Cyclic nucleotide signaling has long been studied for its growth-mitigating properties in the setting of CAD and other vascular disorders. Heme-containing soluble guanylyl cyclase (sGC) synthesizes cyclic guanosine monophosphate (cGMP) and maintains vascular homeostasis predominantly through cGMP-dependent protein kinase (PKG) signaling. Considering that reactive oxygen species (ROS) can interfere with appropriate sGC signaling by oxidizing the cyclase heme moiety and so are associated with several CVD pathologies, the current study was designed to test the hypothesis that heme-independent sGC activation by BAY 60-2770 (BAY60) maintains cGMP levels despite heme oxidation and inhibits ASM cell migration through phosphorylation of the PKG target and actin-binding vasodilator-stimulated phosphoprotein (VASP). First, using the heme oxidant ODQ, cGMP content was potentiated in the presence of BAY60. Using a rat model of arterial growth, BAY60 significantly reduced neointima formation and luminal narrowing compared to vehicle (VEH)-treated controls. In rat ASM cells BAY60 significantly attenuated cell migration, reduced G:F actin, and increased PKG activity and VASP Ser239 phosphorylation (pVASP·S239) compared to VEH controls. Site-directed mutagenesis was then used to generate overexpressing full-length wild type VASP (FL-VASP/WT), VASP Ser239 phosphorylation-mimetic (FL-VASP/239D) and VASP Ser239 phosphorylation-resistant (FL-VASP/239A) ASM cell mutants. Surprisingly, FL-VASP/239D negated the inhibitory effects of FL-VASP/WT and FL-VASP/239A cells on migration. Furthermore, when FL-VASP mutants were treated with BAY60, only the FL-VASP/239D group showed reduced migration compared to its VEH controls. Intriguingly, FL-VASP/239D abrogated the stimulatory effects of FL-VASP/WT and FL-VASP/239A cells on PKG activity. In turn, pharmacologic blockade of PKG in the presence of BAY60 reversed the inhibitory effect of BAY60 on naïve ASM cell migration. Taken together, we demonstrate for the first time that BAY60 inhibits ASM cell migration through cGMP/PKG/VASP signaling yet through mechanisms independent of pVASP·S239 and that FL-VASP overexpression regulates PKG activity in rat ASM cells. These findings implicate BAY60 as a potential pharmacotherapeutic agent against aberrant ASM growth disorders such as CAD and also establish a unique mechanism through which VASP controls PKG activity., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: It is unknown whether keeping a firearm in the home confers protection against crime or, instead, increases the risk of violent crime in the home. To study risk factors for homicide in the home, we identified homicides occurring in the homes of victims in three metropolitan counties., Methods: After each homicide, we obtained data from the police or medical examiner and interviewed a proxy for the victim. The proxies' answers were compared with those of control subjects who were matched to the victims according to neighborhood, sex, race, and age range. Crude and adjusted odds ratios were calculated with matched-pairs methods., Results: During the study period, 1860 homicides occurred in the three counties, 444 of them (23.9 percent) in the home of the victim. After excluding 24 cases for various reasons, we interviewed proxy respondents for 93 percent of the victims. Controls were identified for 99 percent of these, yielding 388 matched pairs. As compared with the controls, the victims more often lived alone or rented their residence. Also, case households more commonly contained an illicit-drug user, a person with prior arrests, or someone who had been hit or hurt in a fight in the home. After controlling for these characteristics, we found that keeping a gun in the home was strongly and independently associated with an increased risk of homicide (adjusted odds ratio, 2.7; 95 percent confidence interval, 1.6 to 4.4). Virtually all of this risk involved homicide by a family member or intimate acquaintance., Conclusions: The use of illicit drugs and a history of physical fights in the home are important risk factors for homicide in the home. Rather than confer protection, guns kept in the home are associated with an increase in the risk of homicide by a family member or intimate acquaintance.

The comprehensive metabolic profile of endogenous opioid peptides is established here for human pituitary for the first time. Sixteen human pituitaries, obtained postmortem, were analyzed individually by gradient reversed-phase high-performance liquid chromatography together with a radio-receptor assay with [3H]etorphine as ligand. This combination was used to detect opioid receptor activity. The 16 assay profiles were sufficiently consistent for a composite of them to serve as a comparative basis for other studies on the pathophysiology of the human pituitary. To demonstrate one selected comparison, we present data on a distinctively different profile of opioid receptor activity in the pituitary of one patient who died from a drug overdose.

We have discovered a 20-day-old infant who possessed anatomic evidence of chronic hypoxemia with right ventricular hypertrophy and who died in hypoxic hypoxemia with a postmortem PO2 of 4 mm. Hg. Subsequently, and ECG was discovered which had been obtained at one day of age and showed Q-T interval prolongation along with T-wave alternation. We believe this case to be one of the first to substantiate the mechanism for SIDS as proposed by Schwartz, 26 with hypoxia acting synergistically with a prolonged Q-T interval causing sudden unexpected death in this infant--providing a link between cardiac and respiratory mechanisms of death.

A 3-week-old infant died of sudden infant death syndrome (crib death, cot death). Although prolongation of the Q-T interval was found on an electrocardiogram taken at birth, this child appeared to have died of terminal respiratory failure as judged by a postmortem arterial oxygen tension of 4 mm Hg. The heart showed right ventricular hypertrophy, a finding consistent with chronic hypoxemia. The prolongation of the Q-T interval may have contributed to the death of this child with hypoxia predisposing to a fatal episode of ventricular fibrillation.