Heterozygous Tropomodulin 3 mice have improved lung vascularization after chronic hypoxia.

The molecular mechanisms leading to high-altitude pulmonary hypertension (HAPH) remains poorly understood. We previously analyzed the whole genome sequence of Kyrgyz highland population and identified eight genomic intervals having a potential role in HAPH. Tropomodulin 3 gene (TMOD3), which encodes a protein that binds and caps the pointed ends of actin filaments and inhibits cell migration, was one of the top candidates. Here we systematically sought additional evidence to validate the functional role of TMOD3. In-silico analysis reveals that some of the SNPs in HAPH associated genomic intervals were positioned in a regulatory region that could result in alternative splicing of TMOD3. In order to functionally validate the role of TMOD3 in HAPH, we exposed Tmod3-/+ mice to 4 weeks of constant hypoxia, i.e. 10% O2 and analyzed both functional (hemodynamic measurements) and structural (angiography) parameters related to HAPH. The hemodynamic measurements, such as right ventricular systolic pressure, a surrogate measure for pulmonary arterial systolic pressure, and right ventricular contractility (RV- ± dP/dt), increases with hypoxia did not separate between Tmod3-/+ and control mice. Remarkably, there was a significant increase in the number of lung vascular branches and total length of pulmonary vascular branches (P < 0.001) in Tmod3-/+ after 4 weeks of constant hypoxia as compared with controls. Notably, the Tmod3-/+ endothelial cells migration was also significantly higher than that from the wild-type littermates. Our results indicate that, under chronic hypoxia, lower levels of Tmod3 play an important role in the maintenance or neo-vascularization of pulmonary arteries.

Multiomic approach and Mendelian randomization analysis identify causal associations between blood biomarkers and subcortical brain structure volumes.

Alterations in subcortical brain structure volumes have been found to be associated with several neurodegenerative and psychiatric disorders. At the same time, genome-wide association studies (GWAS) have identified numerous common variants associated with brain structure. In this study, we integrate these findings, aiming to identify proteins, metabolites, or microbes that have a putative causal association with subcortical brain structure volumes via a two-sample Mendelian randomization approach. This method uses genetic variants as instrument variables to identify potentially causal associations between an exposure and an outcome. The exposure data that we analyzed comprised genetic associations for 2994 plasma proteins, 237 metabolites, and 103 microbial genera. The outcome data included GWAS data for seven subcortical brain structure volumes including accumbens, amygdala, caudate, hippocampus, pallidum, putamen, and thalamus. Eleven proteins and six metabolites were found to have a significant association with subcortical structure volumes, with nine proteins and five metabolites replicated using independent exposure data. We found causal associations between accumbens volume and plasma protease c1 inhibitor as well as strong association between putamen volume and Agouti signaling protein. Among metabolites, urate had the strongest association with thalamic volume. No significant associations were detected between the microbial genera and subcortical brain structure volumes. We also observed significant enrichment for biological processes such as proteolysis, regulation of the endoplasmic reticulum apoptotic signaling pathway, and negative regulation of DNA binding. Our findings provide insights to the mechanisms through which brain volumes may be affected in the pathogenesis of neurodevelopmental and psychiatric disorders and point to potential treatment targets for disorders that are associated with subcortical brain structure volumes.

Mechanosensitive channel Piezo1 is required for pulmonary artery smooth muscle cell proliferation.

Concentric pulmonary vascular wall thickening due partially to increased pulmonary artery (PA) smooth muscle cell (PASMC) proliferation contributes to elevating pulmonary vascular resistance (PVR) in patients with pulmonary hypertension (PH). Although pulmonary vasoconstriction may be an early contributor to increasing PVR, the transition of contractile PASMCs to proliferative PASMCs may play an important role in the development and progression of pulmonary vascular remodeling in PH. A rise in cytosolic Ca2+ concentration ([Ca2+]cyt) is a trigger for PASMC contraction and proliferation. Here, we report that upregulation of Piezo1, a mechanosensitive cation channel, is involved in the contractile-to-proliferative phenotypic transition of PASMCs and potential development of pulmonary vascular remodeling. By comparing freshly isolated PA (contractile PASMCs) and primary cultured PASMCs (from the same rat) in a growth medium (proliferative PASMCs), we found that Piezo1, Notch2/3, and CaSR protein levels were significantly higher in proliferative PASMCs than in contractile PASMCs. Upregulated Piezo1 was associated with an increase in expression of PCNA, a marker for cell proliferation, whereas downregulation (with siRNA) or inhibition (with GsMTx4) of Piezo1 attenuated PASMC proliferation. Furthermore, Piezo1 in the remodeled PA from rats with experimental PH was upregulated compared with PA from control rats. These data indicate that PASMC contractile-to-proliferative phenotypic transition is associated with the transition or adaptation of membrane channels and receptors. Upregulated Piezo1 may play a critical role in PASMC phenotypic transition and PASMC proliferation. Upregulation of Piezo1 in proliferative PASMCs may likely be required to provide sufficient Ca2+ to assure nuclear/cell division and PASMC proliferation, contributing to the development and progression of pulmonary vascular remodeling in PH.

Endothelial upregulation of mechanosensitive channel Piezo1 in pulmonary hypertension.

Piezo is a mechanosensitive cation channel responsible for stretch-mediated Ca2+ and Na+ influx in multiple types of cells. Little is known about the functional role of Piezo1 in the lung vasculature and its potential pathogenic role in pulmonary arterial hypertension (PAH). Pulmonary arterial endothelial cells (PAECs) are constantly under mechanic stretch and shear stress that are sufficient to activate Piezo channels. Here, we report that Piezo1 is significantly upregulated in PAECs from patients with idiopathic PAH and animals with experimental pulmonary hypertension (PH) compared with normal controls. Membrane stretch by decreasing extracellular osmotic pressure or by cyclic stretch (18% CS) increases Ca2+-dependent phosphorylation (p) of AKT and ERK, and subsequently upregulates expression of Notch ligands, Jagged1/2 (Jag-1 and Jag-2), and Delta like-4 (DLL4) in PAECs. siRNA-mediated downregulation of Piezo1 significantly inhibited the stretch-mediated pAKT increase and Jag-1 upregulation, whereas downregulation of AKT by siRNA markedly attenuated the stretch-mediated Jag-1 upregulation in human PAECs. Furthermore, the mRNA and protein expression level of Piezo1 in the isolated pulmonary artery, which mainly contains pulmonary arterial smooth muscle cells (PASMCs), from animals with severe PH was also significantly higher than that from control animals. Intraperitoneal injection of a Piezo1 channel blocker, GsMTx4, ameliorated experimental PH in mice. Taken together, our study suggests that membrane stretch-mediated Ca2+ influx through Piezo1 is an important trigger for pAKT-mediated upregulation of Jag-1 in PAECs. Upregulation of the mechanosensitive channel Piezo1 and the resultant increase in the Notch ligands (Jag-1/2 and DLL4) in PAECs may play a critical pathogenic role in the development of pulmonary vascular remodeling in PAH and PH.

MDM2-Mediated Ubiquitination of Angiotensin-Converting Enzyme 2 Contributes to the Development of Pulmonary Arterial Hypertension.

BACKGROUND: Angiotensin-converting enzyme 2 (ACE2) converts angiotensin II, a potent vasoconstrictor, to angiotensin-(1-7) and is also a membrane protein that enables coronavirus disease 2019 (COVID-19) infectivity. AMP-activated protein kinase (AMPK) phosphorylation of ACE2 enhances ACE2 stability. This mode of posttranslational modification of ACE2 in vascular endothelial cells is causative of a pulmonary hypertension (PH)-protective phenotype. The oncoprotein MDM2 (murine double minute 2) is an E3 ligase that ubiquitinates its substrates to cause their degradation. In this study, we investigated whether MDM2 is involved in the posttranslational modification of ACE2 through its ubiquitination of ACE2, and whether an AMPK and MDM2 crosstalk regulates the pathogenesis of PH. METHODS: Bioinformatic analyses were used to explore E3 ligase that ubiquitinates ACE2. Cultured endothelial cells, mouse models, and specimens from patients with idiopathic pulmonary arterial hypertension were used to investigate the crosstalk between AMPK and MDM2 in regulating ACE2 phosphorylation and ubiquitination in the context of PH. RESULTS: Levels of MDM2 were increased and those of ACE2 decreased in lung tissues or pulmonary arterial endothelial cells from patients with idiopathic pulmonary arterial hypertension and rodent models of experimental PH. MDM2 inhibition by JNJ-165 reversed the SU5416/hypoxia-induced PH in C57BL/6 mice. ACE2-S680L mice (dephosphorylation at S680) showed PH susceptibility, and ectopic expression of ACE2-S680L/K788R (deubiquitination at K788) reduced experimental PH. Moreover, ACE2-K788R overexpression in mice with endothelial cell-specific AMPKα2 knockout mitigated PH. CONCLUSIONS: Maladapted posttranslational modification (phosphorylation and ubiquitination) of ACE2 at Ser-680 and Lys-788 is involved in the pathogenesis of pulmonary arterial hypertension and experimental PH. Thus, a combined intervention of AMPK and MDM2 in the pulmonary endothelium might be therapeutically effective in PH treatment.

TRPC6, a therapeutic target for pulmonary hypertension.

Idiopathic pulmonary arterial hypertension (PAH) is a fatal and progressive disease. Sustained vasoconstriction due to pulmonary arterial smooth muscle cell (PASMC) contraction and concentric arterial remodeling due partially to PASMC proliferation are the major causes for increased pulmonary vascular resistance and increased pulmonary arterial pressure in patients with precapillary pulmonary hypertension (PH) including PAH and PH due to respiratory diseases or hypoxemia. We and others observed upregulation of TRPC6 channels in PASMCs from patients with PAH. A rise in cytosolic Ca2+ concentration ([Ca2+]cyt) in PASMC triggers PASMC contraction and vasoconstriction, while Ca2+-dependent activation of PI3K/AKT/mTOR pathway is a pivotal signaling cascade for cell proliferation and gene expression. Despite evidence supporting a pathological role of TRPC6, no selective and orally bioavailable TRPC6 antagonist has yet been developed and tested for treatment of PAH or PH. In this study, we sought to investigate whether block of receptor-operated Ca2+ channels using a nonselective blocker of cation channels, 2-aminoethyl diphenylborinate (2-APB, administered intraperitoneally) and a selective blocker of TRPC6, BI-749327 (administered orally) can reverse established PH in mice. The results from the study show that intrapulmonary application of 2-APB (40 µM) or BI-749327 (3-10 µM) significantly and reversibly inhibited acute alveolar hypoxia-induced pulmonary vasoconstriction. Intraperitoneal injection of 2-APB (1 mg/kg per day) significantly attenuated the development of PH and partially reversed established PH in mice. Oral gavage of BI-749327 (30 mg/kg, every day, for 2 wk) reversed established PH by ∼50% via regression of pulmonary vascular remodeling. Furthermore, 2-APB and BI-749327 both significantly inhibited PDGF- and serum-mediated phosphorylation of AKT and mTOR in PASMC. In summary, the receptor-operated and mechanosensitive TRPC6 channel is a good target for developing novel treatment for PAH/PH. BI-749327, a selective TRPC6 blocker, is potentially a novel and effective drug for treating PAH and PH due to respiratory diseases or hypoxemia.

Hypoxic vascular response and ventilation/perfusion matching in end-stage COPD may depend on p22phox.

Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease in which the amount of emphysema and airway disease may be very different between individuals, even in end-stage disease. Emphysema formation may be linked to the involvement of the small pulmonary vessels. The NAPDH oxidase (Nox) family is emerging as a key disease-related factor in vascular diseases, but currently its role in hypoxia-induced pulmonary remodelling in COPD remains unclear.Here we investigate the role of p22phox, a regulatory subunit of Nox, in COPD lungs, hypoxic pulmonary vasoconstriction (HPV), hypoxia-induced pulmonary vascular remodelling and pulmonary hypertension.In COPD, compared to control lungs, p22phox expression was significantly reduced. The expression was correlated positively with mean pulmonary arterial pressure and oxygenation index and negatively with the diffusing capacity of the lung for carbon monoxide (p<0.02). This suggests a role of p22phox in ventilation/perfusion ratio matching, vascular remodelling and loss of perfused lung area. In p22phox-/- mice, HPV was significantly impaired. In the chronic hypoxic setting, lack of p22phox was associated with improved right ventricular function and decreased pulmonary vascular remodelling.p22phox-dependent Nox plays an important role in the COPD phenotype, by its action on phase II HPV and chronic vascular remodelling.

Docosahexaenoic acid causes rapid pulmonary arterial relaxation via KCa channel-mediated hyperpolarisation in pulmonary hypertension.

Cardioprotective benefits of ω-3 fatty acids such as docosahexaenoic acid (DHA) are well established, but the regulatory effect of DHA on vascular tone and pressure in pulmonary hypertension is largely unknown.As DHA is a potent regulator of K+ channels, we hypothesised that DHA modulates the membrane potential of pulmonary artery smooth muscle cells (PASMCs) through K+ channels and thus exerts its effects on pulmonary vascular tone and pressure.We show that DHA caused dose-dependent activation of the calcium-activated K+ (KCa) current in primary human PASMCs and endothelium-dependent relaxation of pulmonary arteries. This vasodilation was significantly diminished in KCa-/- (Kcnma1-/-) mice. In vivo, acute DHA returned the right ventricular systolic pressure in the chronic hypoxia-induced pulmonary hypertension animal model to the level of normoxic animals. Interestingly, in idiopathic pulmonary arterial hypertension the KCa channels and their subunits were upregulated. DHA activated KCa channels in these human PASMCs and hyperpolarised the membrane potential of the idiopathic pulmonary arterial hypertension PASMCs to that of the PASMCs from healthy donors.Our findings indicate that DHA activates PASMC KCa channels leading to vasorelaxation in pulmonary hypertension. This effect might provide a molecular explanation for the previously undescribed role of DHA as an acute vasodilator in pulmonary hypertension.

Robot-assisted ureteric reconstructive surgeries for benign diseases: Initial single-center experience with point of technique.

INTRODUCTION: We present our initial experience with robot-assisted reconstructive surgeries with the Da Vinci Xi robotic system for benign ureteric pathologies. MATERIALS AND METHODS: This is a retrospective review of prospectively collected data of patients who underwent robot-assisted reconstructive procedures for benign diseases of the ureter at our department from April 2018 to November 2022. Demographic and perioperative details were recorded. Patients were followed up and surgical success was evaluated on the basis of symptomatic, functional, and radiological improvement. RESULTS: A total of 34 patients underwent robot-assisted reconstructions for benign ureteric pathologies by various techniques. Mean age, body mass index (BMI), hospital stay and follow-up duration were 36 years, 24.1 kg/m2, 5.29 days, and 7.08 months respectively. Procedures included pyeloplasty in eight, primary ureteroneocystostomy (UNC) in seven, Psoas hitch UNC in five, Boari flap UNC in six, Ureteroureterostomy in four, ureterocalicostomy in two and ileal ureteral transposition in two patients. Mean docking time, total operative time, and estimated blood loss were 31.5 min, 178 min, and 64.3 ml, respectively. All patients had radiologic or functional improvement on follow-up after 6 months. CONCLUSION: Robot-assisted reconstructive surgery for benign ureteric and bladder pathologies imparted excellent short-term outcomes without major complications with all the advantages of a minimally invasive approach.

Multiomic approach and Mendelian randomization analysis identify causal associations between blood biomarkers and subcortical brain structure volumes.

Alterations in subcortical brain structure volumes have been found to be associated with several neurodegenerative and psychiatric disorders. At the same time, genome-wide association studies (GWAS) have identified numerous common variants associated with brain structure. In this study, we integrate these findings, aiming to identify proteins, metabolites, or microbes that have a putative causal association with subcortical brain structure volumes via a two-sample Mendelian randomization approach. This method uses genetic variants as instrument variables to identify potentially causal associations between an exposure and an outcome. The exposure data that we analyzed comprised genetic associations for 2,994 plasma proteins, 237 metabolites, and 103 microbial genera. The outcome data included GWAS data for seven subcortical brain structure volumes including accumbens, amygdala, caudate, hippocampus, pallidum, putamen, and thalamus. Eleven proteins and six metabolites were found to have a significant association with subcortical structure volumes. We found causal associations between amygdala volume and granzyme A as well as association between accumbens volume and plasma protease c1 inhibitor. Among metabolites, urate had the strongest association with thalamic volume. No significant associations were detected between the microbial genera and subcortical brain structure volumes. We also observed significant enrichment for biological processes such as proteolysis, regulation of the endoplasmic reticulum apoptotic signaling pathway, and negative regulation of DNA binding. Our findings provide insights to the mechanisms through which brain volumes may be affected in the pathogenesis of neurodevelopmental and psychiatric disorders and point to potential treatment targets for disorders that are associated with subcortical brain structure volumes.