The Melatonin Derivative ITH13001 Prevents Hypertension and Cardiovascular Alterations in Angiotensin II-Infused Mice.

Inflammatory mechanisms and oxidative stress seem to contribute to the pathogenesis of hypertension. ITH13001 is a melatonin-phenyl-acrylate hybrid that moderately induces the antioxidant transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) and has a potent oxidant scavenging effect compared with other derivatives of its family. Here we investigated the effect of ITH13001 on hypertension and the associated cardiovascular alterations. Angiotensin II (AngII)-infused mice were treated with ITH13001 (1 mg/kg per day, i.p.) for 2 weeks. The ITH13001 treatment prevented: 1) the development of hypertension, cardiac hypertrophy, and increased collagen and B-type natriuretic peptide (Bnp) expression in the heart; 2) the reduction of elasticity, incremental distensibility, fenestrae area, intraluminal diameter, and endothelial cell number in mesenteric resistance arteries (MRA); 3) the endothelial dysfunction in aorta and MRA; 4) the plasma and cardiovascular oxidative stress and the reduced aortic nitric oxide (NO) bioavailability; 5) the increased cardiac levels of the cytokines interleukin (IL)-1ß, IL-6, and C-C motif chemokine ligand 2 (Ccl2), the T cell marker cluster of differentiation 3 (Cd3), the inflammasome NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3), the proinflammatory enzymes inducible nitric oxide synthase (iNOS) and COX-2, the toll-like receptor 4 (TLR4) adapter protein myeloid differentiation primary response 88 (MyD88), and the nuclear factor kappa B (NF-κB) subunit p65; 6) the greater aortic expression of the cytokines tumor necrosis factor alpha (Tnf-α), Ccl2 and IL-6, Cd3, iNOS, MyD88, and NLRP3. Although ITH13001 increased nuclear Nrf2 levels and heme oxygenase 1 (HO-1) expression in vascular smooth muscle cells, both cardiac and vascular Nrf2, Ho-1, and NADPH quinone dehydrogenase 1 (Nqo1) levels remained unmodified irrespective of AngII infusion. Summarizing, ITH13001 improved hypertension-associated cardiovascular alterations independently of Nrf2 pathway activation, likely due to its direct antioxidant and anti-inflammatory properties. Therefore, ITH13001 could be a useful therapeutic strategy in patients with resistant hypertension. SIGNIFICANCE STATEMENT: Despite the existing therapeutic arsenal, only half of the patients treated for hypertension have adequately controlled blood pressure; therefore, the search for new compounds to control this pathology and the associated damage to end-target organs (cerebral, cardiac, vascular, renal) is of particular interest. The present study demonstrates that a new melatonin derivative, ITH13001, prevents hypertension development and the associated cardiovascular alterations due to its antioxidant and anti-inflammatory properties, making this compound a potential candidate for treatment of resistant hypertensive patients.

The hemodynamic response to nitrite is acute and dependent upon tissue perfusion.

In the vasculature, nitric oxide (NO) is produced in the endothelium by endothelial nitric oxide synthase (eNOS) and is critical for the regulation of blood flow and blood pressure. Blood flow may also be regulated by the formation of nitrite-derived NO catalyzed by hemoproteins under hypoxic conditions. We sought to investigate whether nitrite administration may affect tissue perfusion and systemic hemodynamics in WT and eNOS knockout mice. We found that global eNOS KO mice show decreased tissue perfusion compared to WT mice by using laser speckle contrast imaging. To study both the acute and long-term effects of sodium nitrite (0, 0.1, 1, and 10 mg/kg) on peripheral blood flow and systemic blood pressure, a bolus of nitrite was delivered intraperitoneally every 24 h over 4 consecutive days. We found that nitrite administration resulted in a dose-dependent and acute increase in peripheral blood flow in eNOS KO mice but had no effects in WT mice. The nitrite induced changes in tissue perfusion were transient, as determined by intraindividual comparisons of tissue perfusion 24-h after injection. Accordingly, 10 mg/kg sodium nitrite acutely decreased blood pressure in eNOS KO mice but not in WT mice as determined by invasive Millar catheterization. Interestingly, we found the vasodilatory effects of nitrite to be inversely correlated to baseline tissue perfusion. These results demonstrate the nitrite acutely recovers hypoperfusion and hypertension in global eNOS KO mice and suggest the vasodilatory actions of nitrite are dependent upon tissue hypoperfusion.

Combined in vivo metabolic effects of quetiapine and methadone in brain and blood of rats.

Changes in pharmacokinetics and endogenous metabolites may underlie additive biological effects of concomitant use of antipsychotics and opioids. In this study, we employed untargeted metabolomics analysis and targeted analysis to examine the changes in drug metabolites and endogenous metabolites in the prefrontal cortex (PFC), midbrain, and blood of rats following acute co-administration of quetiapine and methadone. Rats were divided into four groups and received cumulative increasing doses of quetiapine (QTP), methadone (MTD), quetiapine + methadone (QTP + MTD), or vehicle (control). All samples were analyzed using liquid chromatography-mass spectrometry (LC-MS). Our findings revealed increased levels of the quetiapine metabolites: Norquetiapine, O-dealkylquetiapine, 7-hydroxyquetiapine, and quetiapine sulfoxide, in the blood and brain when methadone was present. Our study also demonstrated a decrease in methadone and its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in the rat brain when quetiapine was present. Despite these findings, there were only small differences in the levels of 225-296 measured endogenous metabolites due to co-administration compared to single administrations. For example, N-methylglutamic acid, glutaric acid, p-hydroxyphenyllactic acid, and corticosterone levels were significantly decreased in the brain of rats treated with both compounds. Accumulation of serotonin in the midbrain was additionally observed in the MTD group, but not in the QTP + MTD group. In conclusion, this study in rats suggests a few but important additive metabolic effects when quetiapine and methadone are co-administered.

NLRP3 activation contributes to endothelin-1-induced erectile dysfunction.

In the present study, we hypothesized that endothelin (ET) receptors (ETA and ETB ) stimulation, through increased calcium and ROS formation, leads to Nucleotide Oligomerization Domain-Like Receptor Family, Pyrin Domain Containing 3 (NLRP3) activation. Intracavernosal pressure (ICP/MAP) was measured in C57BL/6 (WT) mice. Functional and immunoblotting assays were performed in corpora cavernosa (CC) strips from WT, NLRP3-/- and caspase-/- mice in the presence of ET-1 (100 nM) and vehicle, MCC950, tiron, BAPTA AM, BQ123, or BQ788. ET-1 reduced the ICP/MAP in WT mice, and MCC950 prevented the ET-1 effect. ET-1 decreased CC ACh-, sodium nitroprusside (SNP)-induced relaxation, and increased caspase-1 expression. BQ123 an ETA receptor antagonist reversed the effect. The ETB receptor antagonist BQ788 also reversed ET-1 inhibition of ACh and SNP relaxation. Additionally, tiron, BAPTA AM, and NLRP3 genetic deletion prevented the ET-1-induced loss of ACh and SNP relaxation. Moreover, BQ123 diminished CC caspase-1 expression, while BQ788 increased caspase-1 and IL-1ß levels in a concentration-dependent manner (100 nM-10 µM). Furthermore, tiron and BAPTA AM prevented ET-1-induced increase in caspase-1. In addition, BAPTA AM blocked ET-1-induced ROS generation. In conclusion, ET-1-induced erectile dysfunction depends on ETA - and ETB -mediated activation of NLRP3 in mouse CC via Ca2+ -dependent ROS generation.

Impaired phosphate transport in SLC34A2 variants in patients with pulmonary alveolar microlithiasis.

BACKGROUND: Variants in SLC34A2 encoding the sodium-dependent phosphate transport protein 2b (NaPi-IIb) cause the rare lung disease pulmonary alveolar microlithiasis (PAM). PAM is characterised by the deposition of calcium-phosphate concretions in the alveoli usually progressing over time. No effective treatment is available. So far, 30 allelic variants in patients have been reported but only a few have been functionally characterised. This study aimed to determine the impact of selected SLC34A2 variants on transporter expression and phosphate uptake in cellular studies. METHODS: Two nonsense variants (c.910A > T and c.1456C > T), one frameshift (c.1328delT), and one in-frame deletion (c.1402_1404delACC) previously reported in patients with PAM were selected for investigation. Wild-type and mutant c-Myc-tagged human NaPi-IIb constructs were expressed in Xenopus laevis oocytes. The transport function was investigated with a 32Pi uptake assay. NaPi-IIb protein expression and localisation were determined with immunoblotting and immunohistochemistry, respectively. RESULTS: Oocytes injected with the wild-type human NaPi-IIb construct had significant 32Pi transport compared to water-injected oocytes. In addition, the protein had a molecular weight as expected for the glycosylated form, and it was readily detectable in the oocyte membrane. Although the protein from the Thr468del construct was synthesised and expressed in the oocyte membrane, phosphate transport was similar to non-injected control oocytes. All other mutants were non-functional and not expressed in the membrane, consistent with the expected impact of the truncations caused by premature stop codons. CONCLUSIONS: Of four analysed SLC34A2 variants, only the Thr468del showed similar protein expression as the wild-type cotransporter in the oocyte membrane. All mutant transporters were non-functional, supporting that dysfunction of NaPi-IIb underlies the pathology of PAM.

Combined effects of methadone and quetiapine on respiratory rate, haemodynamic variables, and temperature in conscious rats.

Fatal poisonings where both methadone and quetiapine are detected post-mortem occurs frequently in legal autopsy cases. It is unclear whether quetiapine increases the risk of fatal methadone poisoning or if it is merely detected due to widespread use. We hypothesized that methadone and quetiapine would have additive toxic effects on respiratory rate, blood pressure, and the QTc-interval. To investigate this hypothesis, we used telemetry implants for measurements of respiratory rate, haemodynamic variables, the velocity of blood pressure changes, temperature, and movement in conscious, freely moving male Wistar rats aged 12-13 weeks. The combined effects of three accumulative i.p. doses of methadone (2.5, 10, 15 mg/kg) and quetiapine (3, 10, 30 mg/kg) were compared to rats treated with the same doses of each drug alone, and a vehicle-treated group in a randomized investigator blinded study. No additive effects of quetiapine and methadone on respiratory rate, haemodynamic variables, or movement were observed. However, body temperature was significantly lower by approximately 1.5°C on average in the group treated with both methadone and quetiapine (15 + 30 mg/kg) compared to the other groups. This indicates a synergistic effect of quetiapine and methadone on thermoregulation, which may increase the risk of fatal poisoning. We suggest studying this finding further in human settings.

A Systematic Review of the Effect of Vericiguat on Patients with Heart Failure.

Despite recent advances in heart failure (HF) therapy, the risk of cardiovascular (CV) mortality, morbidity, and HF hospitalization (HFH) are major challenges in HF treatment. We aimed to review the potential of vericiguat as a treatment option for HF. A systematic literature review was performed using the PubMed database and ClinicalTrials.gov. Four randomized controlled trials were identified, which study the safety and efficacy of vericiguat in HF patients. Vericiguat activates soluble guanylate cyclase (sGC) by binding to the beta-subunit, bypassing the requirement for NO-induced activation. The nitric oxide (NO)-sGC-cyclic guanosine monophosphate (cGMP) pathway plays an essential role in cardiovascular (CV) regulation and the protection of healthy cardiac function but is impaired in HF. Vericiguat reduced the risk of CV death and HFH in HF patients with reduced ejection fraction (HFrEF) but showed no therapeutic effect on HF with preserved ejection fraction (HFpEF). The trials demonstrated a favorable safety profile with most common adverse events such as hypotension, syncope, and anemia. Therefore, vericiguat is recommended for patients with HFrEF and a minimum systolic blood pressure of 100 mmHg. Treatment with vericiguat is considered when the individual patient experiences decompensation despite being on guideline-recommended medication, e.g., angiotensin-converting inhibitor/AT1 receptor antagonist, beta-adrenoceptor antagonist, spironolactone, and sodium-glucose transporter 2 inhibitors. Furthermore, larger studies are required to investigate any potential effect of vericiguat in HFpEF patients. Despite the limitations, vericiguat can be recommended for patients with HFrEF, where standard-of-care is insufficient, and the disease worsens.

Sex-Dependent Impairment of Endothelium-Dependent Relaxation in Aorta of Mice with Overexpression of Hyaluronan in Tunica Media.

Diabetic macroangiopathy is characterized by increased extracellular matrix deposition, including excessive hyaluronan accumulation, vessel thickening and stiffness, and endothelial dysfunction in large arteries. We hypothesized that the overexpression of hyaluronan in the tunica media also led to endothelial cell (EC) dysfunction. To address this hypothesis, we investigated the following in the aortas of mice with excessive hyaluronan accumulation in the tunica media (HAS-2) and wild-type mice: EC dysfunction via myograph studies, nitric oxide (NO) bioavailability via diaminofluorescence, superoxide formation via dihydroethidium fluorescence, and the distances between ECs via stereological methods. EC dysfunction, characterized by blunted relaxations in response to acetylcholine and decreased NO bioavailability, was found in the aortas of male HAS-2 mice, while it was unaltered in the aortas of female HAS-2 mice. Superoxide levels increased and extracellular superoxide dismutase (ecSOD) expression decreased in the aortas of male and female HAS-2 mice. The EC-EC distances and LDL receptor expression were markedly increased in the HAS-2 aortas of male mice. Our findings suggest hyaluronan increases oxidative stress in the vascular wall and that together with increased EC distance, it is associated with a sex-specific decrease in NO levels and endothelial dysfunction in the aorta of male HAS-2 transgenic mice.

Erectile Dysfunction and Altered Contribution of KCa1.1 and KCa2.3 Channels in the Penile Tissue of Type-2 Diabetic db/db Mice.

BACKGROUND: Activation of endothelial small conductance calcium-activated K+ channels (KCa2.3) and intermediate conductance calcium-activated K+ channels (KCa3.1) leads to vascular relaxation. We found endothelial KCa2.3 down-regulation in the corpus cavernosum diminishes erectile function. AIM: We hypothesized that in type-2 diabetic mice, the function of KCa2.3 and KCa1.1 channels is impaired in erectile tissue. METHODS: Erectile function was measured, and corpus cavernosum strips were mounted for functional studies and processed for qPCR and immunoblotting. OUTCOMES: Effects of type 2 diabetes on erectile function, expression and function of calcium-activated potassium channels. RESULTS: In anesthetized diabetic db/db mice, erectile function was markedly decreased compared to non-diabetic heterozygous db/+ mice, and the impairment was even more pronounced compared to normal C57BL/6 mice. qPCR revealed KCa2.3 and KCa1.1α channel expressions were upregulated in corpus cavernosum from db/db mice. Immunoblotting showed down-regulation of KCa2.3 channels in the corpus cavernosum from db/db mice. Acetylcholine relaxations were impaired while relaxations induced by the nitric oxide, donor SNP were unaltered in corpus cavernosum from db/db compared to C57BL/6 and db/+ mice. Apamin, a blocker of KCa2 channels, inhibited acetylcholine relaxation in corpus cavernosum from all experimental groups. In the presence of apamin, acetylcholine relaxation was markedly decreased in corpus cavernosum from db/db vs C57BL/6 and db/+ mice. An opener of KCa2 and KCa3.1 channels, NS309, potentiated acetylcholine relaxations in corpus cavernosum from db/+ and db/db mice. Iberiotoxin, a blocker of KCa1.1 channels, inhibited acetylcholine relaxation in corpus cavernosum from db/+ mice, while there was no effect in tissue from db/db mice. CLINICAL TRANSLATION: Erectile function in diabetic db/db mice was severely affected compared to heterozygous and control mice, findings suggesting the non-diabetic db/+ and diabetic db/db mice for translational purpose can be used for drug testing on, respectively, moderate and severe erectile dysfunction. The altered expressions and impaired acetylcholine relaxation in the presence of apamin compared to C57BL/6 mice may suggest decreased KCa1.1 channel function may underpin impaired endothelium-dependent relaxation and erectile dysfunction in diabetic db/db mice. STRENGTHS & LIMITATIONS: The present study provides a mouse model for type 2 diabetes to test moderate and severe erectile dysfunction drugs. Decreased KCa1.1 channel function contributes to erectile dysfunction, and it is a limitation that it is not supported by electrophysiological measurements. CONCLUSION: Our results suggest that the contribution of iberiotoxin-sensitive KCa1.1 channels to relaxation is reduced in the corpus cavernosum, while apamin-sensitive KCa2.3 channels appear upregulated. The impaired KCa1.1 channel function may contribute to the impaired erectile function in diabetic db/db mice.

Erectile Dysfunction and Altered Contribution of KCa1.1 and KCa2.3 Channels in the Penile Tissue of Type-2 Diabetic db/db Mice.

BACKGROUND: Activation of endothelial small conductance calcium-activated K+ channels (KCa2.3) and intermediate conductance calcium-activated K+ channels (KCa3.1) leads to vascular relaxation. We found endothelial KCa2.3 down-regulation in the corpus cavernosum diminishes erectile function. AIM: We hypothesized that in type-2 diabetic mice, the function of KCa2.3 and KCa1.1 channels is impaired in erectile tissue. METHODS: Erectile function was measured, and corpus cavernosum strips were mounted for functional studies and processed for qPCR and immunoblotting. OUTCOMES: Effects of type 2 diabetes on erectile function, expression and function of calcium-activated potassium channels. RESULTS: In anesthetized diabetic db/db mice, erectile function was markedly decreased compared to non-diabetic heterozygous db/+ mice, and the impairment was even more pronounced compared to normal C57BL/6 mice. qPCR revealed KCa2.3 and KCa1.1α channel expressions were upregulated in corpus cavernosum from db/db mice. Immunoblotting showed down-regulation of KCa2.3 channels in the corpus cavernosum from db/db mice. Acetylcholine relaxations were impaired while relaxations induced by the nitric oxide, donor SNP were unaltered in corpus cavernosum from db/db compared to C57BL/6 and db/+ mice. Apamin, a blocker of KCa2 channels, inhibited acetylcholine relaxation in corpus cavernosum from all experimental groups. In the presence of apamin, acetylcholine relaxation was markedly decreased in corpus cavernosum from db/db vs C57BL/6 and db/+ mice. An opener of KCa2 and KCa3.1 channels, NS309, potentiated acetylcholine relaxations in corpus cavernosum from db/+ and db/db mice. Iberiotoxin, a blocker of KCa1.1 channels, inhibited acetylcholine relaxation in corpus cavernosum from db/+ mice, while there was no effect in tissue from db/db mice. CLINICAL TRANSLATION: Erectile function in diabetic db/db mice was severely affected compared to heterozygous and control mice, findings suggesting the non-diabetic db/+ and diabetic db/db mice for translational purpose can be used for drug testing on, respectively, moderate and severe erectile dysfunction. The altered expressions and impaired acetylcholine relaxation in the presence of apamin compared to C57BL/6 mice may suggest decreased KCa1.1 channel function may underpin impaired endothelium-dependent relaxation and erectile dysfunction in diabetic db/db mice. STRENGTHS & LIMITATIONS: The present study provides a mouse model for type 2 diabetes to test moderate and severe erectile dysfunction drugs. Decreased KCa1.1 channel function contributes to erectile dysfunction, and it is a limitation that it is not supported by electrophysiological measurements. CONCLUSION: Our results suggest that the contribution of iberiotoxin-sensitive KCa1.1 channels to relaxation is reduced in the corpus cavernosum, while apamin-sensitive KCa2.3 channels appear upregulated. The impaired KCa1.1 channel function may contribute to the impaired erectile function in diabetic db/db mice. Comerma-Steffensen S, Prat-Duran J, Mogensen S, et al. Erectile Dysfunction and Altered Contribution of KCa1.1 and KCa2.3 Channels in the Penile Tissue of Type-2 Diabetic db/db Mice. J Sex Med 2022;19:697-710.

Combined effects of quetiapine and opioids: A study of autopsy cases, drug users and sedation in rats.

Fatal opioid poisonings often involve methadone or morphine. This study aimed to elucidate if quetiapine, a widely used sedative antipsychotic medication, may increase the risk of fatal opioid poisoning by additive inhibitory effects on the central nervous system. We used data from 323 cases of fatal methadone or/and morphine poisonings autopsied from 2013 to 2020, a survey of 34 drug users, and performed blinded placebo-controlled studies in 75 Flinders Resistant Line rats receiving three cumulative intraperitoneal doses of vehicle, methadone (2.5, 10 and 15 mg/kg), morphine (3.75, 15 and 22.5 mg/kg), quetiapine (3, 10 and 30 mg/kg) or quetiapine combined with methadone or morphine. Quetiapine was detected in 20.4% of fatal opioid poisonings with a significantly increased frequency over time, primarily in low or therapeutic concentrations, and was not associated with methadone or morphine concentrations. Use of quetiapine, most commonly in low-to-moderate doses to obtain a sleep-inducing or tranquillizing effect, was reported by 67.6% of survey respondents. In the animal studies, a significant impairment of sedation score, performance on the rotarod and open field mobility was observed in all treatment groups compared with vehicle. However, the effect of quetiapine plus the opioid was not significantly different from that of the opioid alone. Thus, no additive sedative effects were observed in rats. Our results suggest that quetiapine is more often an innocent bystander than a contributor to fatal opioid poisoning. However, the combined effects on other parameters, including blood pressure, cardiac rhythm and respiratory rate, need investigation.

Senicapoc treatment in COVID-19 patients with severe respiratory insufficiency-A randomized, open-label, phase II trial.

BACKGROUND: The aim of the current study was to determine if treatment with senicapoc, improves the PaO2 /FiO2 ratio in patients with COVID-19 and severe respiratory insufficiency. METHODS: Investigator-initiated, randomized, open-label, phase II trial in four intensive care units (ICU) in Denmark. We included patients aged ≥18 years and admitted to an ICU with severe respiratory insufficiency due to COVID-19. The intervention consisted of 50 mg enteral senicapoc administered as soon as possible after randomization and again after 24 h. Patients in the control group received standard care only. The primary outcome was the PaO2 /FiO2 ratio at 72 h. RESULTS: Twenty patients were randomized to senicapoc and 26 patients to standard care. Important differences existed in patient characteristics at baseline, including more patients being on non-invasive/invasive ventilation in the control group (54% vs. 35%). The median senicapoc concentration at 72 h was 62.1 ng/ml (IQR 46.7-71.2). The primary outcome, PaO2 /FiO2 ratio at 72 h, was significantly lower in the senicapoc group (mean 19.5 kPa, SD 6.6) than in the control group (mean 24.4 kPa, SD 9.2) (mean difference -5.1 kPa [95% CI -10.2, -0.04] p = .05). The 28-day mortality in the senicapoc group was 2/20 (10%) compared with 6/26 (23%) in the control group (OR 0.36 95% CI 0.06-2.07, p = .26). CONCLUSIONS: Treatment with senicapoc resulted in a significantly lower PaO2 /FiO2 ratio at 72 h with no differences for other outcomes.

The role of SOX family members in solid tumours and metastasis.

Cancer is a heavy burden for humans across the world with high morbidity and mortality. Transcription factors including sex determining region Y (SRY)-related high-mobility group (HMG) box (SOX) proteins are thought to be involved in the regulation of specific biological processes. The deregulation of gene expression programs can lead to cancer development. Here, we review the role of the SOX family in breast cancer, prostate cancer, renal cell carcinoma, thyroid cancer, brain tumours, gastrointestinal and lung tumours as well as the entailing therapeutic implications. The SOX family consists of more than 20 members that mediate DNA binding by the HMG domain and have regulatory functions in development, cell-fate decision, and differentiation. SOX2, SOX4, SOX5, SOX8, SOX9, and SOX18 are up-regulated in different cancer types and have been found to be associated with poor prognosis, while the up-regulation of SOX11 and SOX30 appears to be favourable for the outcome in other cancer types. SOX2, SOX4, SOX5 and other SOX members are involved in tumorigenesis, e.g. SOX2 is markedly up-regulated in chemotherapy resistant cells. The SoxF family (SOX7, SOX17, SOX18) plays an important role in angio- and lymphangiogenesis, with SOX18 seemingly being an attractive target for anti-angiogenic therapy and the treatment of metastatic disease in cancer. In summary, SOX transcription factors play an important role in cancer progression, including tumorigenesis, changes in the tumour microenvironment, and metastasis. Certain SOX proteins are potential molecular markers for cancer prognosis and putative potential therapeutic targets, but further investigations are required to understand their physiological functions.

Cystamine Treatment Fails to Prevent the Development of Pulmonary Hypertension in Chronic Hypoxic Rats.

INTRODUCTION: Pulmonary hypertension is characterized by vasoconstriction and remodeling of pulmonary arteries, leading to right ventricular hypertrophy and failure. We have previously found upregulation of transglutaminase 2 (TG2) in the right ventricle of chronic hypoxic rats. The hypothesis of the present study was that treatment with the transglutaminase inhibitor, cystamine, would inhibit the development of pulmonary arterial remodeling, pulmonary hypertension, and right ventricular hypertrophy. METHODS: Effect of cystamine on transamidase activity was investigated in tissue homogenates. Wistar rats were exposed to chronic hypoxia and treated with vehicle, cystamine (40 mg/kg/day in mini-osmotic pumps), sildenafil (25 mg/kg/day), or the combination for 2 weeks. RESULTS: Cystamine concentration-dependently inhibited TG2 transamidase activity in liver and lung homogenates. In contrast to cystamine, sildenafil reduced right ventricular systolic pressure and hypertrophy and decreased pulmonary vascular resistance and muscularization in chronic hypoxic rats. Fibrosis in the lung tissue decreased in chronic hypoxic rats treated with cystamine. TG2 expression was similar in the right ventricle and lung tissue of drug and vehicle-treated hypoxic rats. DISCUSSION/CONCLUSIONS: Cystamine inhibited TG2 transamidase activity, but cystamine failed to prevent pulmonary hypertension, right ventricular hypertrophy, and pulmonary arterial muscularization in the chronic hypoxic rat.

Eight novel variants in the SLC34A2 gene in pulmonary alveolar microlithiasis.

BACKGROUND: Pulmonary alveolar microlithiasis (PAM) is caused by genetic variants in the SLC34A2 gene, which encodes the sodium-dependent phosphate transport protein 2B (NaPi-2b). PAM is characterised by deposition of calcium phosphate concretions (microliths) in the alveoli leading to pulmonary dysfunction. The variant spectrum of SLC34A2 has not been well investigated and it is not yet known whether a genotype-phenotype correlation exists. METHODS: We collected DNA from 14 patients with PAM and four relatives, and analysed the coding regions of SLC34A2 by direct DNA sequencing. To determine the phenotype characteristics, clinical data were collected and a severity score was created for each variant, based on type and localisation within the protein. RESULTS: We identified eight novel allelic variants of SLC34A2 in 14 patients with PAM. Four of these were nonsense variants, three were missense and one was a splice site variant. One patient was heterozygous for two different variants and all other patients were homozygous. Four patients were asymptomatic and 10 patients were symptomatic. The severity of the disease was associated with the variant severity. CONCLUSIONS: Our findings support a significant role for SLC34A2 in PAM and expand the variant spectrum of the disease. Thus, SLC34A2 variants were detected in all patients and eight novel allelic variants were discovered. An association between disease severity and the severity of the variants was found; however, this needs to be investigated in larger patient populations.

Terlipressin Increases Systemic and Lowers Pulmonary Arterial Pressure in Experimental Acute Pulmonary Embolism.

OBJECTIVES: We investigated whether the vasopressin-analog, terlipressin induces systemic vasoconstriction and pulmonary vasodilation in a porcine model of acute pulmonary embolism. DESIGN: Controlled, animal study. SETTING: Tertiary medical center research laboratory. SUBJECTS: Female pigs (n = 12, Cross of Land Race, Duroc, and Yorkshire ~ 60 kg). INTERVENTIONS: Acute pulmonary embolism was induced by administration of three large autologous emboli. Animals then received four increasing doses of either terlipressin (n = 6) or vehicle (n = 6). MEASUREMENTS AND MAIN RESULTS: Effects were evaluated in vivo at baseline, after pulmonary embolism and after each dose by invasive hemodynamic measures, transesophageal echocardiography, and blood analysis. Isolated pulmonary arteries were evaluated ex vivo in a myograph. Pulmonary embolism caused a four-fold increase in pulmonary vascular resistance (p < 0.0001) and a two-fold increase in mean pulmonary arterial pressure (p < 0.0001) compared with baseline. Terlipressin increased mean systemic blood pressure (28 ± 5 mm Hg; p < 0.0001) and systemic vascular resistance (1,320 ± 143 dynes; p < 0.0001) compared with vehicle. In the pulmonary circulation, terlipressin decreased mean pulmonary arterial pressure (-6.5 ± 1.8 mm Hg; p = 0.005) and tended to decrease pulmonary vascular resistance (-83 ± 33 dynes; p = 0.07). Terlipressin decreased cardiac output (-2.5 ± 0.5 L/min; p < 0.0001) and increased plasma lactate (2.7 ± 0.2 mmol/L; p < 0.0001), possibly indicating systemic hypoperfusion. A biomarker of cerebral ischemia, S100b, remained unchanged, suggesting preserved cerebral perfusion (0.17 ± 0.11 µg/L; p = 0.51). Ex vivo, terlipressin relaxed pulmonary and constricted mesenteric arteries. CONCLUSIONS: Terlipressin caused systemic vasoconstriction and pulmonary vasodilation in a porcine in vivo model of acute pulmonary embolism and vasorelaxation in isolated pulmonary arteries. Despite positive vascular effects, cardiac output declined and plasma lactate increased probably due to a predominantly systemic vasoconstrictor effect of terlipressin. These findings should warrant careful translation to the clinical setting and does not suggest routine use in acute pulmonary embolism.

Cholinergic regulation along the pulmonary arterial tree of the South American rattlesnake: vascular reactivity, muscarinic receptors, and vagal innervation.

Vascular tone in the reptilian pulmonary vasculature is primarily under cholinergic, muscarinic control exerted via the vagus nerve. This control has been ascribed to a sphincter located at the arterial outflow, but we speculated whether the vascular control in the pulmonary artery is more widespread, such that responses to acetylcholine and electrical stimulation, as well as the expression of muscarinic receptors, are prevalent along its length. Working on the South American rattlesnake (Crotalus durissus), we studied four different portions of the pulmonary artery (truncus, proximal, distal, and branches). Acetylcholine elicited robust vasoconstriction in the proximal, distal, and branch portions, but the truncus vasodilated. Electrical field stimulation (EFS) caused contractions in all segments, an effect partially blocked by atropine. We identified all five subtypes of muscarinic receptors (M1-M5). The expression of the M1 receptor was largest in the distal end and branches of the pulmonary artery, whereas expression of the muscarinic M3 receptor was markedly larger in the truncus of the pulmonary artery. Application of the neural tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate (DiI) revealed widespread innervation along the whole pulmonary artery, and retrograde transport of the same tracer indicated two separate locations in the brainstem providing vagal innervation of the pulmonary artery, the medial dorsal motor nucleus of the vagus and a ventro-lateral location, possibly constituting a nucleus ambiguus. These results revealed parasympathetic innervation of a large portion of the pulmonary artery, which is responsible for regulation of vascular conductance in C. durissus, and implied its integration with cardiorespiratory control.

Impact of pulmonary endarterectomy on pulmonary arterial wave propagation and reservoir function.

High wave speed and large wave reflection in the pulmonary artery have previously been reported in patients with chronic thromboembolic pulmonary hypertension (CTEPH). We assessed the impact of pulmonary endarterectomy (PEA) on pulmonary arterial wave propagation and reservoir function in patients with CTEPH. Right heart catheterization was performed using a combined pressure and Doppler flow sensor-tipped guidewire to obtain simultaneous pressure and flow velocity measurements in the pulmonary artery in eight patients with CTEPH before and 3 mo after PEA. Wave intensity and reservoir-excess pressure analyses were then performed. Following PEA, mean pulmonary arterial pressure (PAPm; ∼49 vs. ∼32 mmHg), pulmonary vascular resistance (PVR; ∼11.1 vs. ∼5.1 Wood units), and wave speed (∼16.5 vs. ∼8.1 m/s), i.e., local arterial stiffness, markedly decreased. The changes in the intensity of the reflected arterial wave and wave reflection index (pre: ∼28%; post: ∼22%) were small, and patients post-PEA with and without residual pulmonary hypertension (i.e., PAPm ≥ 25 mmHg) had similar wave reflection index (∼20 vs. ∼23%). The reservoir and excess pressure decreased post-PEA, and the changes were associated with improved right ventricular afterload, function, and size. In conclusion, although PVR and arterial stiffness decreased substantially following PEA, large wave reflection persisted, even in patients without residual pulmonary hypertension, indicating lack of improvement in vascular impedance mismatch. This may continue to affect the optimal ventriculoarterial interaction, and further studies are warranted to determine whether this contributes to persistent symptoms in some patients.NEW & NOTEWORTHY We performed wave intensity analysis in the pulmonary artery in patients with chronic thromboembolic pulmonary hypertension before and 3 mo after pulmonary endarterectomy. Despite substantial reduction in pulmonary arterial pressures, vascular resistance, and arterial stiffness, large pulmonary arterial wave reflection persisted 3 mo postsurgery, even in patients without residual pulmonary hypertension, suggestive of lack of improvement in vascular impedance mismatch.

Extracellular l-arginine Enhances Relaxations Induced by Opening of Calcium-Activated SKCa Channels in Porcine Retinal Arteriole.

We investigated whether the substrate for nitric oxide (NO) production, extracellular l-arginine, contributes to relaxations induced by activating small (SKCa) conductance Ca2+-activated potassium channels. In endothelial cells, acetylcholine increased 3H-l-arginine uptake, while blocking the SKCa and the intermediate (IKCa) conductance Ca2+-activated potassium channels reduced l-arginine uptake. A blocker of the y+ transporter system, l-lysine also blocked 3H-l-arginine uptake. Immunostaining showed co-localization of endothelial NO synthase (eNOS), SKCa3, and the cationic amino acid transporter (CAT-1) protein of the y+ transporter system in the endothelium. An opener of SKCa channels, cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA) induced large currents in endothelial cells, and concentration-dependently relaxed porcine retinal arterioles. In the presence of l-arginine, concentration-response curves for CyPPA were leftward shifted, an effect unaltered in the presence of low sodium, but blocked by l-lysine in the retinal arterioles. Our findings suggest that SKCa channel activity regulates l-arginine uptake through the y+ transporter system, and we propose that in vasculature affected by endothelial dysfunction, l-arginine administration requires the targeting of additional mechanisms such as SKCa channels to restore endothelium-dependent vasodilatation.

Impact of chronic hypoxia on proximal pulmonary artery wave propagation and mechanical properties in rats.

Arterial stiffness and wave reflection are important components of the ventricular afterload. Therefore, we aimed to assess the arterial wave characteristics and mechanical properties of the proximal pulmonary arteries (PAs) in the hypoxic pulmonary hypertensive rat model. After 21 days in normoxic or hypoxic chambers (24 animals/group), animals underwent transthoracic echocardiography and PA catheterization with a dual-tipped pressure and Doppler flow sensor wire. Wave intensity analysis was performed. Artery rings obtained from the pulmonary trunk, right and left PAs, and aorta were subjected to a tensile test to rupture. Collagen and elastin content were determined. In hypoxic rats, proximal PA wall thickness, collagen content, tensile strength per unit collagen, maximal elastic modulus, and wall viscosity increased, whereas the elastin-to-collagen ratio and arterial distensibility decreased. Arterial pulse wave velocity was also increased, and the increase was more prominent in vivo than ex vivo. Wave intensity was similar in hypoxic and normoxic animals with negligible wave reflection. In contrast, the aortic maximal elastic modulus remained unchanged, whereas wall viscosity decreased. In conclusion, there was no evidence of altered arterial wave propagation in proximal PAs of hypoxic rats while the extracellular matrix protein composition was altered and collagen tensile strength increased. This was accompanied by altered mechanical properties in vivo and ex vivo. NEW & NOTEWORTHY In rats exposed to chronic hypoxia, we have shown that pulse wave velocity in the proximal pulmonary arteries increased and pressure dependence of the pulse wave velocity was steeper in vivo than ex vivo leading to a more prominent increase in vivo.