Longevity biotechnology: bridging AI, biomarkers, geroscience and clinical applications for healthy longevity.

The recent unprecedented progress in ageing research and drug discovery brings together fundamental research and clinical applications to advance the goal of promoting healthy longevity in the human population. We, from the gathering at the Aging Research and Drug Discovery Meeting in 2023, summarised the latest developments in healthspan biotechnology, with a particular emphasis on artificial intelligence (AI), biomarkers and clocks, geroscience, and clinical trials and interventions for healthy longevity. Moreover, we provide an overview of academic research and the biotech industry focused on targeting ageing as the root of age-related diseases to combat multimorbidity and extend healthspan. We propose that the integration of generative AI, cutting-edge biological technology, and longevity medicine is essential for extending the productive and healthy human lifespan.

Pharmacological modulation of transglutaminase 2 in the unilateral ureteral obstruction mouse model.

BACKGROUND: Transglutaminase 2 (TG2) is a multifunctional enzyme involved in fibrosis by promoting transforming-growth-factor-ß1 and crosslinking of extracellular matrix proteins. These functions are dependent on the open conformation, while the closed state of TG2 can induce vasodilation. We explored the putative protective role of TG2 in its closed state on development of renal fibrosis and blood pressure (BP) regulation. METHODS: We studied the unilateral ureteral obstruction (UUO) mouse model treated with LDN27219, which promotes the closed conformation of TG2. Mice were subjected to 7 days UUO or sham operation and treated with vehicle (n=10), LDN27219 (15 mg/kg/12 hours, n=9) or candesartan (5 mg/kg/day, n=10) as a clinically comparator. Renal expression of TG2 and pro-fibrotic mediators were evaluated by Western blotting, qPCR and histology, and BP by tail-cuff measurements. RESULTS: Obstructed kidneys showed increased mRNA and protein expression of fibronectin, collagen 3α1 (Col3α1), α-smooth muscle actin and collagen staining. Despite increased renal TG2 mRNA, protein expression was reduced in all UUO groups, but with increased transamidase activity in the vehicle and candesartan groups. LDN27219 reduced mRNA expression of fibronectin and Col3α1, but their protein expression remained unchanged. In contrast to LDN27219, candesartan lowered BP without affecting expression of pro-fibrotic biomarkers. CONCLUSION: Renal TG2 mRNA and protein expression levels seem dissociated, with transamidase activity being increased. LDN27219 influences kidney pro-fibrotic markers at the mRNA level and attenuates transamidase activity but without affecting collagen content or BP. Our findings suggest that TG2 in its closed conformation has anti-fibrotic effects at the molecular level.

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.

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.

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.

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.

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.

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.

Differential contribution of renal cytochrome P450 enzymes to kidney endothelial dysfunction and vascular oxidative stress in obesity.

Arachidonic acid (AA)-derived cytochrome P450 (CYP) derivatives, epoxyeicosatrienoic acids (EETs) and 20-hidroxyeicosatetranoic acid (20-HETE), play a key role in kidney tubular and vascular functions and blood pressure. Altered metabolism of CYP epoxygenases and CYP hydroxylases has differentially been involved in the pathogenesis of metabolic disease-associated vascular complications, although the mechanisms responsible for the vascular injury are unclear. The present study aimed to assess whether obesity-induced changes in CYP enzymes may contribute to oxidative stress and endothelial dysfunction in kidney preglomerular arteries. Endothelial function and reactive oxygen species (ROS) production were assessed in interlobar arteries of obese Zucker rats (OZR) and their lean counterparts lean Zucker rats (LZR) and the effects of CYP2C and CYP4A inhibitors sulfaphenazole and HET0016, respectively, were examined on the endothelium-dependent relaxations and O2- and H2O2 levels of preglomerular arteries. Non-nitric oxide (NO) non-prostanoid endothelium-derived hyperpolarization (EDH)-type responses were preserved but resistant to the CYP epoxygenase blocker sulfaphenazole in OZR in contrast to those in LZR. Sulfaphenazole did not further inhibit reduced arterial H2O2 levels, and CYP2C11/CYP2C23 enzymes were downregulated in intrarenal arteries from OZR. Renal EDH-mediated relaxations were preserved in obese rats by the enhanced activity and expression of endothelial calcium-activated potassium channels (KCa). CYP4A blockade restored impaired NO-mediated dilatation and inhibited augmented O2- production in kidney arteries from OZR. The current data demonstrate that both decreased endothelial CYP2C11/ CYP2C23-derived vasodilator H2O2 and augmented CYP4A-derived 20-HETE contribute to endothelial dysfunction and vascular oxidative stress in obesity. CYP4A inhibitors ameliorate arterial oxidative stress and restore endothelial function which suggests its therapeutic potential for the vascular complications of obesity-associated kidney injury.

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.

Transglutaminase 2 Inhibitor LDN 27219 Age-Dependently Lowers Blood Pressure and Improves Endothelium-Dependent Vasodilation in Resistance Arteries.

Transglutaminase 2 (TG2) is an enzyme which in the open conformation exerts transamidase activity, leading to protein cross-linking and fibrosis. In the closed conformation, TG2 participates in transmembrane signaling as a G protein. The unspecific transglutaminase inhibitor cystamine causes vasorelaxation in rat resistance arteries. However, the role of TG2 conformation in vascular function is unknown. We investigated the vascular effects of selective TG2 inhibitors by myography in isolated rat mesenteric and human subcutaneous resistance arteries, patch-clamp studies on vascular smooth muscle cells, and blood pressure measurements in rats and mice. LDN 27219 promoted the closed TG2 conformation and inhibited transamidase activity in mesenteric arteries. In contrast to TG2 inhibitors promoting the open conformation (Z-DON, VA5), LDN 27219 concentration-dependently relaxed rat and resistance human arteries by a mechanism dependent on nitric oxide, large-conductance calcium-activated and voltage-gated potassium channels 7, lowering blood pressure. LDN 27219 also potentiated acetylcholine-induced relaxation by opening potassium channels in the smooth muscle; these effects were abolished by membrane-permeable TG2 inhibitors promoting the open conformation. In isolated arteries from 35- to 40-week-old rats, transamidase activity was increased, and LDN 27219 improved acetylcholine-induced relaxation more than in younger rats. Infusion of LDN 27219 decreased blood pressure more effectively in 35- to 40-week than 12- to 14-week-old anesthetized rats. In summary, pharmacological modulation of TG2 to the closed conformation age-dependently lowers blood pressure and, by opening potassium channels, potentiates endothelium-dependent vasorelaxation. Our findings suggest that promoting the closed conformation of TG2 is a potential strategy to treat age-related vascular dysfunction and lowers blood pressure.

Transglutaminase 2 as a novel target in chronic kidney disease - Methods, mechanisms and pharmacological inhibition.

Chronic kidney disease (CKD) is a global health problem with a prevalence of 10-15%. Progressive fibrosis of the renal tissue is a main feature of CKD, but current treatment strategies are relatively unspecific and delay, but do not prevent, CKD. Exploration of novel pharmacological targets to inhibit fibrosis development are therefore important. Transglutaminase 2 (TG2) is known to be central for extracellular collagenous matrix formation, but TG2 is a multifunctional enzyme and novel research has broadened our view on its extra- and intracellular actions. TG2 exists in two conformational states with different catalytic properties as determined by substrate availability and local calcium concentrations. The open conformation of TG2 depends on calcium and has transamidase activity, central for protein modification and cross-linking of extracellular protein components, while the closed conformation is a GTPase involved in transmembrane signaling processes. We first describe different methodologies to assess TG2 activity in renal tissue and cell cultures such as biotin cadaverine incorporation. Then we systematically review animal CKD models and preliminary studies in humans (with diabetic, IgA- and chronic allograft nephropathy) to reveal the role of TG2 in renal fibrosis. Mechanisms behind TG2 activation, TG2 externalization dependent on Syndecan-4 and interactions between TG and profibrotic molecules including transforming growth factor ß and the angiotensin II receptor are discussed. Pharmacological TG2 inhibition shows antifibrotic effects in CKD. However, the translation of TG2 inhibition to treat CKD in patients is a challenge as clinical information is limited, and further studies on pharmacokinetics and efficacy of the individual compounds are required.

Endothelial KCa 1.1 and KCa 3.1 channels mediate rat intrarenal artery endothelium-derived hyperpolarization response.

AIM: Endothelium-derived hyperpolarization (EDH)-mediated response plays an essential role in the control of kidney preglomerular circulation, but the identity of the K+ channels involved in this response is still controversial. We hypothesized that large- (KCa 1.1), intermediate- (KCa 3.1) and small (KCa 2.3) -conductance Ca2+ -activated K+ (KCa ) channels are expressed in the endothelium of the preglomerular circulation and participate in the EDH-mediated response. METHODS: We study the functional expression of different K+ channels in non-cultured, freshly isolated native endothelial cells (ECs) of rat intrarenal arteries using immunofluorescence and the patch-clamp technique. We correlate this with vasorelaxant responses ex vivo using wire myography. RESULTS: Immunofluorescence revealed the expression of KCa 1.1, KCa 3.1 and KCa 2.3 channels in ECs. Under voltage-clamp conditions, acetylcholine induced a marked increase in the outward currents in these cells, sensitive to the blockade of KCa 1.1, KCa 3.1 and KCa 2.3 channels respectively. Isometric myography experiments, under conditions of endothelial nitric oxide synthase and cyclooxygenase inhibition, showed that blockade either of KCa 1.1 or KCa 3.1 channels was able to reduce the endothelium-derived vasorelaxation of isolated interlobar arteries, while their combined blockade completely abolished it. In contrast, blockade of KCa 2.3 channels did not reduce this vasorelaxant response, despite being functionally expressed in the endothelial cells. CONCLUSION: This study shows that KCa 1.1 and KCa 3.1 channels are functionally expressed at the renal vascular endothelium and play a central role in the EDH-mediated relaxation of kidney preglomerular arteries, which is important in the control of renal blood flow and glomerular filtration rate.

Endothelial Dysfunction and Passive Changes in the Aorta and Coronary Arteries of Diabetic db/db Mice.

Endothelial cell dysfunction and vessel stiffening are associated with a worsened prognosis in diabetic patients with cardiovascular diseases. The present study hypothesized that sex impacts endothelial dysfunction and structural changes in arteries from diabetic mice. In diabetic (db/db) and normoglycaemic (db/db+) mice, the mechanical properties were investigated in pressurized isolated left anterior descending coronary arteries and aorta segments that were subjected to tensile testing. Functional studies were performed on wire-mounted vascular segments. The male and female db/db mice were hyperglycaemic and had markedly increased body weight. In isolated aorta segments without the contribution of smooth muscle cells, load to rupture, viscoelasticity, and collagen content were decreased suggesting larger distensibility of the arterial wall in both male and female db/db mice. In male db/db aorta segments with smooth muscle cell contribution, lumen diameter was smaller and the passive stretch-tension curve was leftward-shifted, while they were unaltered in female db/db aorta segments versus control db/db+ mice. In contrast to female db/db mice, coronary arteries from male db/db mice had altered stress-strain relationships and increased distensibility. Transthoracic echocardiography revealed a dilated left ventricle with unaltered cardiac output, while aortic flow velocity was decreased in male db/db mice. Impairment of acetylcholine relaxation was aggravated in aorta from female db/db compared to control and male db/db mice, while impairment of sodium nitroprusside relaxations was only observed in aorta from male db/db mice. The remodeling in the coronary arteries and aorta suggests an adaptation of the arterial wall to the reduced flow velocity with sex-specific differences in the passive properties of aorta and coronary arteries. The findings of less distensible arteries and more pronounced endothelial dysfunction in female compared to male diabetic mice may have implications for the observed higher incidence of macrovascular complications in diabetic women.

Pirfenidone Is a Vasodilator: Involvement of KV7 Channels in the Effect on Endothelium-Dependent Vasodilatation in Type-2 Diabetic Mice.

Endothelial cell dysfunction and fibrosis are associated with worsening of the prognosis in patients with cardiovascular disease. Pirfenidone has a direct antifibrotic effect, but vasodilatation may also contribute to the effects of pirfenidone. Therefore, in a first study we investigated the mechanisms involved in the relaxant effect of pirfenidone in rat intrapulmonary arteries and coronary arteries from normal mice. Then in a second study, we investigated whether pirfenidone restores endothelial function in the aorta and mesenteric arteries from diabetic animals. From 16-18-week old normal male C57BL/6 mice and normoglycemic (db/db+), and type 2 diabetic (db/db) male and female mice, arteries were mounted in microvascular isometric myographs for functional studies, and immunoblotting was performed. In rat pulmonary arteries and mouse coronary arteries, pirfenidone induced relaxations, which were inhibited in preparations without endothelium. In mouse coronary arteries, pirfenidone relaxation was inhibited in the presence of a nitric oxide (NO) synthase inhibitor, NG-nitro-l-arginine (L-NOARG), a blocker of large-conductance calcium-activated potassium channels (BKCa), iberiotoxin, and a blocker of KV7 channels, XE991. Patch clamp studies in vascular smooth muscle revealed pirfenidone increased iberiotoxin-sensitive current. In the aorta and mesenteric small arteries from diabetic db/db mice relaxations induced by the endothelium-dependent vasodilator, acetylcholine, were markedly reduced compared to db/db + mice. Pirfenidone enhanced the relaxations induced by acetylcholine in the aorta from diabetic male and female db/db mice. An opener of KV7 channels, flupirtine, had the same effect as pirfenidone. XE991 reduced the effect of pirfenidone and flupirtine and further reduced acetylcholine relaxations in the aorta. In the presence of iberiotoxin, pirfenidone still increased acetylcholine relaxation in aorta from db/db mice. Immunoblotting for KV7.4, KV7.5, and BKCa channel subunits were unaltered in aorta from db/db mice. Pirfenidone failed to improve acetylcholine relaxation in mesenteric arteries, and neither changed acetylcholine-induced transient decreases in blood pressure in db/db+ and db/db mice. In conclusion, pirfenidone vasodilates pulmonary and coronary arteries. In coronary arteries from normal mice, pirfenidone induces NO-dependent vasodilatation involving BKCa and KV7 channels. Pirfenidone improves endothelium-dependent vasodilatation in aorta from diabetic animals by a mechanism involving voltage-gated KV7 channels, a mechanism that may contribute to the antifibrotic effect of pirfenidone.

Novel selective PDE type 1 inhibitors cause vasodilatation and lower blood pressure in rats.

BACKGROUND AND PURPOSE: The PDE enzymes (PDE1-11) hydrolyse and thus inactivate cyclic nucleotides and are important in the regulation of the cardiovascular system. Here,we have investigated the effects on the cardiovascular system, of two novel selective PDE1 inhibitors, Lu AF41228 and Lu AF58027. EXPERIMENTAL APPROACH: We used rat mesenteric small arteries (internal diameters of 200-300 µm), RT-PCR and measured isometric wall tension. Effects of Lu AF41228 and Lu AF58027 on heart rate and BP were assessed in both anaesthetized and conscious male rats. KEY RESULTS: Nanomolar concentrations of Lu AF41228 and Lu AF58027 inhibited PDE1A, PDE1B and PDE1C enzyme activity, while micromolar concentrations were required to observe inhibitory effects at other PDEs. RT-PCR revealed expression of PDE1A, PDE1B and PDE1C in rat brain, heart and aorta, but only PDE1A and PDE1B in mesenteric arteries. In rat isolated mesenteric arteries contracted with phenylephrine or U46619, Lu AF41228 and Lu AF58027 induced concentration-dependent relaxations which were markedly reduced by inhibitors of guanylate cyclase, ODQ, and adenylate cyclase, SQ22536, and in preparations without endothelium. In anaesthetized rats, Lu AF41228 and Lu AF58027 dose-dependently lowered mean BP and increased heart rate. In conscious rats with telemetric pressure transducers, repeated dosing with Lu AF41228 lowered mean arterial BP 10-15 mmHg and increased heart rate. CONCLUSIONS AND IMPLICATIONS: These novel PDE1 inhibitors induce vasodilation and lower BP, suggesting a potential use of these vasodilators in the treatment of hypertension and vasospasm.

CYP epoxygenase-derived H2O2 is involved in the endothelium-derived hyperpolarization (EDH) and relaxation of intrarenal arteries.

Reactive oxygen species (ROS) like hydrogen peroxide (H2O2) are involved in the in endothelium-derived hyperpolarization (EDH)-type relaxant responses of coronary and mesenteric arterioles. The role of ROS in kidney vascular function has mainly been investigated in the context of harmful ROS generation associated to kidney disease. The present study was sought to investigate whether H2O2 is involved in the endothelium-dependent relaxations of intrarenal arteries as well the possible endothelial sources of ROS generation involved in these responses. Under conditions of cyclooxygenase (COX) and nitric oxide (NO) synthase inhibition, acetylcholine (ACh) induced relaxations and stimulated H2O2 release that were reduced by catalase and by the glutathione peroxidase (GPx) mimetic ebselen in rat renal interlobar arteries, suggesting the involvement of H2O2 in the endothelium-dependent responses. ACh relaxations were also blunted by the CYP2C inhibitor sulfaphenazole and by the NADPH oxidase inhibitor apocynin. Acetylcholine stimulated both superoxide (O2•-) and H2O2 production that were reduced by sulfaphenazole and apocynin. Expression of the antioxidant enzyme CuZnSOD and of the H2O2 reducing enzymes catalase and GPx-1 was found in both intrarenal arteries and renal cortex. On the other hand, exogenous H2O2 relaxed renal arteries by decreasing vascular smooth muscle (VSM) intracellular calcium concentration [Ca2+]i and markedly enhanced endothelial KCa currents in freshly isolated renal endothelial cells. CYP2C11 and CYP2C23 epoxygenases were highly expressed in interlobar renal arteries and renal cortex, respectively, and were co-localized with eNOS in renal endothelial cells. These results demonstrate that H2O2 is involved in the EDH-type relaxant responses of renal arteries and that CYP 2C epoxygenases are physiologically relevant endothelial sources of vasodilator H2O2 in the kidney.

Vascular Reactivity Profile of Novel KCa 3.1-Selective Positive-Gating Modulators in the Coronary Vascular Bed.

Opening of intermediate-conductance calcium-activated potassium channels (KC a 3.1) produces membrane hyperpolarization in the vascular endothelium. Here, we studied the ability of two new KC a 3.1-selective positive-gating modulators, SKA-111 and SKA-121, to (1) evoke porcine endothelial cell KC a 3.1 membrane hyperpolarization, (2) induce endothelium-dependent and, particularly, endothelium-derived hyperpolarization (EDH)-type relaxation in porcine coronary arteries (PCA) and (3) influence coronary artery tone in isolated rat hearts. In whole-cell patch-clamp experiments on endothelial cells of PCA (PCAEC), KC a currents evoked by bradykinin (BK) were potentiated ≈7-fold by either SKA-111 or SKA-121 (both at 1 µM) and were blocked by a KC a 3.1 blocker, TRAM-34. In membrane potential measurements, SKA-111 and SKA-121 augmented bradykinin-induced hyperpolarization. Isometric tension measurements in large- and small-calibre PCA showed that SKA-111 and SKA-121 potentiated endothelium-dependent relaxation with intact NO synthesis and EDH-type relaxation to BK by ≈2-fold. Potentiation of the BK response was prevented by KC a 3.1 inhibition. In Langendorff-perfused rat hearts, SKA-111 potentiated coronary vasodilation elicited by BK. In conclusion, our data show that positive-gating modulation of KC a 3.1 channels improves BK-induced membrane hyperpolarization and endothelium-dependent relaxation in small and large PCA as well as in the coronary circulation of rats. Positive-gating modulators of KC a 3.1 could be therapeutically useful to improve coronary blood flow and counteract impaired coronary endothelial dysfunction in cardiovascular disease.

Involvement of transglutaminase 2 and voltage-gated potassium channels in cystamine vasodilatation in rat mesenteric small arteries.

BACKGROUND AND PURPOSE: Vasodilatation may contribute to the neuroprotective and vascular anti-remodelling effect of the tissue transglutaminase 2 (TG2) inhibitor cystamine. Here, we hypothesized that inhibition of TG2 followed by blockade of smooth muscle calcium entry and/or inhibition of Rho kinase underlies cystamine vasodilatation. EXPERIMENTAL APPROACH: We used rat mesenteric small arteries and RT-PCR, immunoblotting, and measurements of isometric wall tension, intracellular Ca(2+) ([Ca(2+)]i ), K(+) currents (patch clamp), and phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and myosin regulatory light chain, in our experiments. KEY RESULTS: RT-PCR and immunoblotting revealed expression of TG2 in mesenteric small arteries. Cystamine concentration-dependently inhibited responses to phenylephrine, 5-HT and U46619 and for extracellular potassium. Selective inhibitors of TG2, LDN 27129 and T101, also inhibited phenylephrine contraction. An inhibitor of PLC suppressed cystamine relaxation. Cystamine relaxed and reduced [Ca(2+)]i in phenylephrine-contracted arteries. In potassium-contracted arteries, cystamine induced less relaxation without changing [Ca(2+)]i , and these relaxations were blocked by mitochondrial complex inhibitors. Blockers of Kv 7 channels, XE991 and linopirdine, inhibited cystamine relaxation and increases in voltage-dependent smooth muscle currents. Cystamine and the Rho kinase inhibitor Y27632 reduced basal MYPT1-Thr(855) phosphorylation, but only Y27632 reduced phenylephrine-induced increases in MYPT1-Thr(855) and myosin regulatory light chain phosphorylation. CONCLUSIONS AND IMPLICATIONS: Cystamine induced vasodilatation by inhibition of receptor-coupled TG2, leading to opening of Kv channels and reduction of intracellular calcium, and by activation of a pathway sensitive to inhibitors of the mitochondrial complexes I and III. Both pathways may contribute to the antihypertensive and neuroprotective effect of cystamine.