Nitric Oxide Synthase Inhibitor Research Articles

The alarmin, interleukin-33, increases vascular tone via extracellular signal regulated kinase-mediated Ca2+ sensitization and endothelial nitric oxide synthase inhibition.

Alarmins are classified by their release from damaged or ruptured cells. Many alarmins have been found to increase vascular tone and oppose endothelium-dependent dilatation (EDD). Interleukin (IL)-33 plays a prominent role in lung injury and can be released during vascular injury and in chronic studies found to be cardioprotective. Our recent work has implicated IL-33 in acute vascular dysfunction following inhalation of engineered nanomaterials (ENM). However, the mechanisms linking IL-33 to vascular tone have not been interrogated. We therefore aimed to determine whether IL-33 directly influenced microvascular tone and endothelial function. Isolated feed arteries and in vivo arterioles from male and female Sprague-Dawley rats were used to determine direct vascular actions of IL-33. Mesenteric feed arteries and arterioles demonstrated reduced intraluminal diameters when treated with increasing concentrations of recombinant IL-33. IL-33 activated extracellular signal regulated kinase (ERK)1/2 of rat aortic smooth muscle cells but not phosphorylation of myosin light chain kinase. This suggested IL-33may sensitize arterioles to Ca2+-mediated responses. Indeed, IL-33 augmented the myogenic- and phenylephrine-induced vasoconstriction. Additionally, incubation of arterioles with 1ng IL-33 attenuated ACh-mediated EDD. Mechanistically, in human aortic endothelial cells, we demonstrate that IL-33-mediated ERK1/2 activation leads to inhibitory phosphorylation of serine 602 on endothelial nitric oxide synthase. Finally, we demonstrate that IL-33-ERK1/2 contributes to vascular tone following two known inducers of IL-33; ENM inhalation and the rupture endothelial cells. The present study provides novel evidence that IL-33 increases vascular tone via canonical ERK1/2 activation in microvascular smooth muscle and endothelium. Altogether, it is suggested IL-33 plays a critical role in microvascular homeostasis following barrier cell injury. KEY POINTS: Interleukin (IL)-33 causes a concentration-dependent reduction in feed artery diameter. IL-33 acts on vascular smooth muscle cells to augment Ca2+-mediated processes. IL-33 causes inhibitory phosphorylation of endothelial nitric oxide synthase and opposes endothelium-dependent dilatation. Engineered nanomaterial-induced lung injury and endothelial cell rupture in part act through IL-33 to mediate increased vascular tone.

Partial involvement of nitric oxide synthase in increased pilocarpine-induced sweating in exercise-trained men.

The physiological mechanisms involved in augmented cholinergic agonist-induced sweating in exercise-trained individuals remain unclear. This study hypothesizes that nitric oxide synthase (NOS) contributes to augmented pilocarpine-induced sweating in habitually exercise-trained individuals. Endurance-trained and untrained men (n=15 each) iontophoretically received 1% L-NAME, a NOS inhibitor, and saline (control) in the forearm and then administered 0.001% and 1% pilocarpine to evaluate sweat rate. L-NAME administration attenuated pilocarpine-induced sweating by 10% in the exercise-trained (P = 0.004) but not in untrained (P = 0.764) groups independent of pilocarpine concentrations. Results indicate that NOS partially contributes to increased cholinergic sweating in exercise-trained men.

Medicinal Chemistry Insights in Neuronal Nitric Oxide Synthase Inhibitors Containing Nitrogen Heterocyclic Compounds: A Mini Review.

Many scientific reports over the last two decades have focused on the discovery and development of novel nNOS inhibitors. The structural identity of isoforms, bioavailability, pharmacokinetic, and safety profile issues remain major obstacles in the discovery of more potent and selective nNOS inhibitors. This review aims to provide an in-depth overview of the molecular interaction patterns between nNOS active site and inhibitors containing structurally diverse nitrogen heterocyclic compounds and highlight the structural properties needed to develop selective nNOS inhibitors. Previously published data allowed the usage of the structure-driven approach in the designing of selective nNOS inhibitors, which relies on the specific structural features required to achieve isoform-selectivity towards nNOS. The incorporation of chiral pyrrolidine ring, two aminopyridine heads, or a specific amino tail group, along with the inhibitor's capacity to adopt the curled conformation in the nNOS environment significantly strengthens the molecular interaction between the inhibitor and nNOS residues by forming specific electrostatic interactions and non-bonded contacts that are vital for isoform selectivity. Additional structure-activity relationship investigations are necessary to elucidate more structural characteristics that will ultimately resolve the exact structural basis required for isoform-selective inhibition of nNOS.

The expression of contextual fear conditioning involves the dorsal hippocampus TRPV1 receptor interacting with the NMDA/NO/cGMP signalling pathway.

The dorsal hippocampus (dHIP) is pivotal for learning, memory, and defensive responses. Transient receptor potential vanilloid type 1 (TRPV1) receptors in the dHIP modulate contextual fear conditioning by triggering a cascade involving glutamate release, nitric oxide (NO) formation and cyclic guanosine monophosphate (cGMP) production. The present study investigated the involvement of dHIP TRPV1 receptors and their interaction with the glutamate/NO/cGMP signalling pathway in modulating the expression of contextual fear conditioning (CFC). Male Wistar rats were submitted to an aversive contextual conditioning session and, 48 h later, were re-introduced to the same aversive environment where the freezing response and autonomic activity (evidenced by increased arterial pressure and heart rate and a decrease in tail temperature) were measured. The results demonstrated that the TRPV1 antagonist 6-I-CPS in dHIP reduced the expression of CFC, whereas the agonist capsaicin had the opposite effect. Furthermore, dHIP pre-treatment with an NMDA receptor antagonist (AP7), neuronal NO synthase inhibitor (N-propyl-L-arginine), NO scavenger (c-PTIO) or guanylate cyclase inhibitor (ODQ) attenuated capsaicin-induced increases in CFC. Finally, we observed that re-exposure to the aversive chamber increased dHIP NO levels in conditioned animals compared with a non-conditioned group, which was prevented by the administration of the TRPV1 antagonist, 6-I-CPS. Our study revealed that TRPV1 receptors in the dHIP play a crucial role in modulating contextual fear expression by acting through the NMDA receptor/NO/cGMP signalling pathway, providing important insights into the underlying mechanisms and potential therapeutic avenues associated with these pathways.

Abstract 4141446: Sodium Glucose Co-transporter 2 (SGLT2) Inhibitors Promote Resiliency to High Pressure Stress in the Human Microvasculature

Emerging evidence suggests that vascular stress from cardiovascular-related co-morbidities promotes microvascular dysfunction, a key component in the development of heart failure with preserved ejection fraction. The sodium glucose co-transporter 2 (SGLT2) inhibitor empagliflozin has been shown to reduce both morbidity and mortality associated with heart failure with preserved ejection fraction, however the full scope of influence of this therapy on human microvascular function remains unknown. We hypothesized that pre-treatment of isolated human microvessels with empagliflozin will prevent stress-induced endothelial dysfunction as evidenced by preserving both the magnitude of flow-induced dilation (FID) as well as the ability to dilate to nitric oxide. Human resistance arterioles (80-250µm) from healthy adults (defined as patients with ≤1 risk factor for cardiovascular disease) were dissected from discarded surgical adipose tissue and treated with empagliflozin (1µM), or vehicle control (ethanol) for 16-20 hours prior to the flow experiment. Vessels were cannulated for videomicroscopy and subjected to high intraluminal pressure (150mmHg, 30 min), an acute stress known to induce endothelial dysfunction. Vessels were pre-constricted with endothelin-1 prior to initiation of flow. A nonlinear logistic regression was used to determine differences between curves. Compared to vehicle control, vessels pre-treated with empagliflozin (1µM ) exhibited nitric oxide-dependent FID as dilation was impaired in the presence of the nitric oxide synthase inhibitor L-NAME (EC50 Control: 10.7 vs L-NAME 83.45, p=0.0107). This data suggests that empagliflozin, an SGLT2 inhibitor, promotes microvascular resilience to stress via preservation of nitric oxide-mediated FID. The ability to elicit stress resilience may explain in part some of the cardiovascular benefits associated with SGLT2 inhibitors and may offer unique opportunities for early intervention or prevention of microvascular dysfunction associated with comorbidities that contribute to heart failure with preserved ejection fraction.

Resveratrol relieves myocardial ischemia–reperfusion injury through inhibiting AKT nitration modification

ABSTRACT Objective The aim of this study was to clarify whether Protein kinase B (PKB)/AKT is nitrated in myocardial ischemia and reperfusion injury (MIRI) resveratrol (RSV)’s protective effect during this process. Methods We blocked blood flow of the left coronary artery (LAD) of mice and used H9c2 cells under an oxygen-glucose deprivation (OGD) environment as animal and cell models of MIRI. N-methyl-D-aspartic acid receptor (NMDAR) inhibitor MK801, neuronal nitric oxide synthase (nNOS) inhibitor 7-NI and RSV were used as interventions. Nitration of proteins, infarction area, cardiomyocyte apoptosis and AKT nitration sites were detected during this study. Results During in-vivo study, AKT nitration was induced through the NMDAR/nNOS/peroxynitrite (ONOO–) pathway, leading to decreased phosphorylation of AKT and increased cardiomyocyte apoptosis. AKT nitration was decreased and phosphorylation was elevated when administrated with RSV, MK801 and 7-NI. In in-vitro study, AKT nitration and TUNEL positive cells was elevated when administrated with NO donor H9c2 cells after OGD/R, when administrated with RSV, MK801 and 7-NI, AKT nitration and apoptosis was deceased in H9c2 cells. Mass spectrometry revealed that nitration sites of AKT included 14 Tyrosine residues. Discussion RSV could inhibit AKT nitration and elevated phosphorylation through suppressing NMDAR/nNOS/ONOO– pathway and further reduce the apoptosis of cardiomyocytes in of myocardial I/R.

Quantification and interpretation of nitric oxide-dependent cutaneous vasodilation during local heating.

Human cutaneous microdialysis approaches for assessing nitric oxide (NO)-dependent blood flow include local heating (LH) of the skin until a plateau is reached, followed by infusion of a NO synthase inhibitor such as NG-nitro-l-arginine methyl ester (l-NAME); however, varied methods of quantifying and expressing NO-dependent vasodilation can obfuscate data interpretation and reproducibility. We retrospectively assessed NO-dependent vasodilation during LH to 39°C or 42°C, calculated as the 1) absolute contribution of the NO-dependent component (along with baseline and the non-NO-dependent component) to the total cutaneous vascular conductance (CVC) response to LH, normalized to maximal CVC (%CVCmax); 2) difference in %CVCmax (Δ%CVCmax) between the LH plateau and post-NO synthase inhibition (l-NAME plateau; Δ%CVCmax = LH plateau - l-NAME plateau); 3) percentage of the LH plateau attributable to Δ%CVCmax (%plateau = Δ%CVCmax/LH plateau × 100); and 4) %plateau when correcting for baseline. The LH plateaus during 39°C and 42°C were 48 ± 17%CVCmax (9 ± 5% baseline; 2 ± 4% non-NO dependent; 36 ± 15% NO dependent) and 88 ± 10%CVCmax (15 ± 8% baseline; 9 ± 10% non-NO dependent; 64 ± 13% NO dependent), respectively. The absolute contributions of the non-NO-dependent and NO-dependent components of the response (P < 0.0001) and the Δ%CVCmax (66 ± 14 vs. 38 ± 15%) were greater during 42°C compared with 39°C (all P ≤ 0.02); however, there were no differences between the two protocols in %plateau (75 ± 13 vs. 80 ± 10%; P = 0.57) or %plateauBL (88 ± 14 vs. 95 ± 8%; P = 0.31). For both protocols, the values were greater for %plateauBL versus Δ%CVCmax and %plateau (P ≤ 0.0001), and for %plateau versus Δ%CVCmax (P ≤ 0.05). Quantification of NO-dependent skin vasodilation responses to LH is dependent upon the mathematical approach and verbal description, which can meaningfully impact data interpretation and reproducibility.NEW & NOTEWORTHY Local heating protocols are commonly used in conjunction with intradermal microdialysis for assessing nitric oxide (NO)-dependent microvascular function in humans, but various methods used to quantify and describe NO-dependent vasodilation may impact data interpretation. We compared four approaches for quantifying NO-dependent cutaneous vasodilation during local heating at 39°C and 42°C. We identify discrepancies in calculated NO-dependent dilation responses that are dependent upon the mathematical approach and meaningfully impact data interpretation and reproducibility.

The effect of CD38 inhibition in age-associated vascular dysfunction

Abstract Introduction Aging is characterized by age-related vascular dysfunction, mainly caused by endothelial dysfunction and arterial stiffness. Levels of nicotinamide adenine dinucleotide (NAD) decrease with aging and accordingly, NAD is thought to be involved in the progression of age-related vascular dysfunction. Recent studies reported the involvement of CD38 in NAD regulation; indeed, its inhibition significantly prolongs the lifespan of progeroid and aged mice. However, whether CD38 is involved in the progression of age-related vascular dysfunction remains to be investigated. Methods CD38 expression was investigated in aortic homogenates of young (12-16 weeks) and aged (18-24 months) C57BL/6 wild-type mice, as well as in vitro in young (passage 5-7) and senescent (passage 14-15) primary human aortic endothelial cells (HAECs). Ex vivo tension myograph experiments were performed on thoracic aortas isolated from aged C57BL/6 mice (22-24 months old) pre-incubated for 1 hour with CD38-specific inhibitor, 78c, or DMSO as control. The effect of CD38 inhibition was also observed in vitro in senescent HAECs. Results A significant upregulation of CD38 expression was detected in the aortas of aged mice compared to the young ones as well as in senescent HAECs compared to young cells. Ex vivo tension myograph experiments showed a significant increase in endothelium-dependent relaxation responses to acetylcholine upon CD38 inhibition. The improvement of endothelial function was prevented by the addition of endothelial nitric oxide synthase (eNOS) inhibitor (L-NAME), but not by the COXs inhibitor, indomethacin, indicating that the observed effect achieved by CD38 inhibition is eNOS dependent. The involvement of the eNOS pathway was further supported by the in vitro finding in HAECs of increased eNOS activation upon CD38 inhibition. Conclusion The herein-reported data suggests an involvement of CD38 in the development of age-related endothelial dysfunction. Further studies are ongoing to investigate the underlying molecular mechanisms as well as the effect of long-term CD38 inhibitor supplementation on the progression of age-related vascular dysfunction.

Photobiomodulation mitigates diastolic dysfunction, reduces perivascular fibrosis and inflammation, and enhances mitochondrial metabolism and angiogenesis in a mouse model of HFpEF

Abstract Introduction Emerging molecular evidence implicates mitochondrial dysfunction, systemic inflammation, and capillary rarefaction as pivotal contributors to the pathophysiology of HFpEF. Photobiomodulation (PBM) has garnered attention for its potential to augment mitochondrial metabolism, attenuate inflammation, and foster angiogenesis. This investigation aims to delineate the effects of PBM on the pathophysiology of HFpEF in a murine model. Methods To induce HFpEF, 3-month-old male C57BL/6 mice were subjected to a '2-hit model' involving a high-fat diet combined with the vasoconstrictor nitric oxide synthase inhibitor, N(G)-Nitro-L-arginine methyl ester (L-NAME). PBM therapy was administered transdermally over the cardiac region thrice weekly for a duration of 10 weeks using a 904nm super-pulsed laser, with each session lasting 90 seconds and delivering an energy density of 4.2J/cm^2 per diode to a cohort of 12 mice. A sham group of 13 mice underwent identical procedures with the laser deactivated. An additional control group of 10 mice was maintained on a standard diet without any interventions. Echocardiographic assessments were performed at the outset, 7th and 10th week time. Following the 10-week period, mice were euthanized, their hearts harvested and serum withdrawn for further analysis. Results Echocardiography of PBM-treated animals demonstrated significant attenuation of deterioration of diastolic parameters (E’) compared to the sham-treated control group (E’[mm/sec], baseline-vs-10w: PBM, -32.1±5.9-vs- -27.9±6.4; sham, -32.9±8.6-vs- -22.5±5.7; p=0.037). Histological analysis of the cardiac tissue involving picrosirius red staining for collagen revealed that the HFpEF model induced a significant increase in the standardized adventitial collagen compared to controls, which was attenuated by PBM (control 3.0±1.6; HFpEF sham: 3.9±1.8; PBM: 3.4±1.9, p=0.008 control vs sham, p=0.055 PBM vs control; p=0.396 control vs PBM). Multiplex analysis of HFpEF mice showed significantly higher levels of inflammatory chemokines than controls that were attenuated by PBM (mean±SD log[pg/ml] of the chemokine (C-X-C motif) ligand-1 (CXCL-1): PBM=2.52±0.29, Control=2.14±0.35; monocyte chemotactic protein (MCP-1): PBM=0.87±0.46, HFpEF=1.5±0.58, Control=0.8±0.6; p&amp;lt;0.001 and p=0.003 respectively). RNA sequencing revealed 551 genes with significant differential expression (DE) between PBM and Controls (adjusted p-value [padj]&amp;lt;0.1). Ingenuity Pathway Analysis (IPA) analysis revealed that PBM upregulated genes pertaining to mitochondrial metabolism and down-regulated genes active in mitochondrial dysfunction (qPCR [GAPDH] 2–∆∆Ct fold change PBM/Sham: Ndufv2=3.7, Atp5md=3.5, Dnhd1=0.5). Relevant DE genes were validated by quantitative real-time PCR (qPCR). Conclusions PBM attenuated Diastolic dysfunction, fibrosis, pro-inflammatory chemokine secretion and upregulates mitochondrial metabolism in a mouse model of HFpEF.Study designResults

Balance of Antioxidants vs. Oxidants in Perinatal Asphyxia

Perinatal asphyxia refers to an acute event of cerebral ischemia and hypoxia during the perinatal period, leading to various degrees of brain injury. The mechanisms involved in perinatal asphyxia include the production of reactive oxygen species (ROS), accumulation of intracellular calcium, lipid peroxidation, excitatory amino acid receptor overactivation, energy failure, and caspase-mediated cell death. Both primary and secondary neuronal damage are caused by the overproduction of ROS following a hypoxic/ischemic event. ROS can react with nearly any type of molecule, including lipids, proteins, polysaccharides, and DNA. Neonates who suffer from perinatal asphyxia are prone to oxidative stress, which is characterized by a disruption in the oxidant/antioxidant balance, favoring oxidants over the intracellular and extracellular antioxidant scavenging mechanisms. Current research has focused on developing treatment strategies that potentially improve the endogenous antioxidant neuroprotective mechanisms or minimize injury resulting from hypoxia/ischemia. In this narrative review, we aim to present evidence regarding the contribution of oxidant/antioxidant balance to the pathogenesis and progression of perinatal asphyxia. Also, we aim to explore the role of potential antioxidant therapies as promising treatment strategies for perinatal asphyxia, especially as an adjunct to therapeutic hypothermia in infants with perinatal asphyxia. The current literature on antioxidant treatments in newborns is limited; however, allopurinol, melatonin, and erythropoietin have shown some positive effects in clinical trials. Inhibitors of nitric oxide synthase, N-acetylcysteine, and docosahexaenoic acid have shown promising neuroprotective effects in preclinical studies. Finally, nanotherapeutics could potentially modulate oxidative stress in hypoxemic/ischemic brain injury by targeted medication delivery. Future research on neuroprotectants and their processes is warranted to develop innovative treatments for hypoxia/ischemia in clinical practice.

Nitric Oxide-Dependent Regulation of Oxygen-Related Processes in a Rat Model of Lead Neurotoxicity: Influence of the Hypoxia Resistance Factor

Background/Aims: Lead exposure is known to induce oxidative stress and neurotoxicity. Nitric oxide (NO) plays an important role in modulating oxidative stress, with L-arginine as a precursor of NO and Nω-nitro-L-arginine (L-NNA) as an inhibitor of NO synthase, an enzyme that catalyses the production of nitric oxide (NO) from L-arginine. Methods: This study investigated the differential effects of L-arginine and L-NNA on markers of oxidative stress and biochemical changes in brain tissue from rats with different levels of resistance to hypoxia exposed to lead nitrate. Rats with either low or high resistance to hypoxia were exposed to lead nitrate (oral 3.6 mg lead nitrate/kg b.w. per day for 30 days) and treated with L-arginine (600 mg/kg b.w., i.p., 30 min before and after exposure to lead nitrate) or L-NNA (35 mg/kg b.w., i.p., 30 min before and after exposure to lead nitrate). Brain tissue samples were analysed for lipid peroxidation, oxidative modification of proteins, and activity of antioxidant enzymes, including superoxide dismutase, catalase, glutathione reductase, and peroxidase, and total antioxidant status (TAS). We also examined the biomarkers of biochemical pathways involving the activity of alanine and aspartate aminotransferases, succinate dehydrogenase (SDH), and α-ketoglutarate dehydrogenase (KGDH). In addition, the trend observed was supported by assessments of the acetylcholine levels and acetylcholinesterase activity (ACh-AChE system) in brain tissue. Results: In rats with low resistance to hypoxia, the L-arginine treatment significantly reduced lipid peroxidation and oxidative protein modification but increased antioxidant enzyme activity, suggesting a protective effect against lead-induced oxidative stress. Conversely, in rats with high resistance to hypoxia, L-NNA had a protective effect, reducing lead-induced oxidative damage and decreasing lipid peroxidation, whereas L-arginine exacerbated oxidative stress and impaired antioxidant defences. These findings were supported by corresponding changes in the acetylcholine-acetylcholinesterase system, reflecting the observed patterns of lead-induced oxidative stress and neurotoxicity. The study shows that L-arginine exerts a protective effect by reducing lead-induced oxidative damage via an improvement in TAS. Our study shows that lead nitrate exposure significantly increases ala-nine and aspartate aminotransferase activity in brain tissue, with L-arginine exacerbating and L-NNA reversing this effect. The lead nitrate exposure also affected the activities of SDH and KGDH, which are important for cellular energy production and hypoxia resistance, with L-arginine altering SDH activity depending on the level of resistance and L-NNA enhancing both SDH and KGDH activities. These trends were further validated by alterations in the ACh-AChE system, highlighting the differential role of NO-dependent mechanisms in modulating lead-induced neurotoxicity based on hypoxia resistance. Conclusion: These findings suggest potential targeted therapeutic strategies based on the oxidative stress profile and highlight the potential of nitric oxide system modulators in counteracting lead-induced biochemical alterations and the dynamics of the ACh-AChE system depending on the individual physiological reactivity of organisms.

Anti-Inflammatory Activity of Labdane and Norlabdane Diterpenoids from Leonurus sibiricus Related to Modulation of MAPKs Signaling Pathway.

Leonurus sibiricus, a widely cultivated herbaceous plant in Asian countries, exhibits diverse medicinal applications. Recent studies emphasize its pharmacological properties and efficacy in promoting bone health. Besides the known compounds and their pharmacological activities, in this study, we isolated and elucidated two new labdane-type diterpenoids, (3R,5R,6S,10S)-3,6-dihydroxy-15-ethoxy-7-oxolabden-8(9),13(14)-dien-15,16-olide (1) and (4R,5R,10S)-18-hydroxy-14,15-bisnorlabda-8-en-7,13-dione (2), a new natural phenolic compound, and a known compound from L. sibiricus using advanced spectroscopic techniques, including circular dichroism spectroscopy, high-resolution mass spectrometry, and 1- and 2-dimensional NMR. Among these, compound 1 demonstrated potent inhibition of nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) mRNA expression levels, followed by compound 2. Whereas compounds 3 and 4 do not exhibit effectiveness in RAW264.7 macrophages. Moreover, compound 1 suppressed pro-inflammatory markers induced by lipopolysaccharide (LPS) stimulation. Compound 1 also suppressed iNOS and cyclooxygenase-2 (COX-2) protein levels and downregulated pro-inflammatory cytokines. Additionally, compound 1 showed inhibition of the phosphorylation of p38, JNK, and ERK, key mediators of the MAPK signaling pathway. These findings indicate that a natural-derived product, compound 1, might be a potential candidate as an anti-inflammation mediator.

Characterization of human placental fetal vessels in gestational diabetes mellitus.

Gestational diabetes mellitus is one of the most common complications during pregnancy. Its prevalence is rapidly increasing worldwide. Gestational diabetes mellitus is leading to an elevated risk for the development of endothelial dysfunction and cardiovascular diseases both in the mother and the child in later life. The underlying pathophysiological mechanisms are not well-understood. Therefore, we aimed to characterize the endothelial function in fetal placental vessels from mothers with gestational diabetes mellitus. In this study, we distinguished between insulin-treated and diet-controlled gestational diabetes mothers and compared them to a normoglycemic control group. The clinical data confirmed pre-conceptional overweight as a risk factor in women with insulin-treated gestational diabetes mellitus. The insulin-treated gestational diabetes group was also characterized by a recent family history of diabetes compared to mothers of the control or diet-controlled gestational diabetes group. Analyses of blood serum from umbilical cords suggested a reduced fetal insulin metabolism in the insulin-treated gestational diabetes group. Vascular function analysis in fetal placental vessels revealed an altered substance P-induced vasorelaxation in vessels from patients with insulin-dependent gestational diabetes. Inhibition of nitric oxide synthase affected only fetal vessel segments from the control group or diet-controlled gestational diabetes group, but not from insulin-dependent gestational diabetes. Finally, we found a significantly decreased substance P receptor (TACR1) mRNA expression in fetal vessel segments from patients with insulin-treated gestational diabetes. In conclusion, we provide evidence that different pathophysiological mechanisms might be responsible for the development of insulin-treated versus diet-controlled gestational diabetes. Only in fetal vessels from patients with insulin-treated gestational diabetes were we able to detect an endothelial dysfunction and a reduced fetal insulin conversion. This provides novel insights into the pathophysiology of the subtypes of gestational diabetes.

Nitric Oxide Signaling and Sensing in Age-Related Diseases.

Nitric oxide (NO) is a key signaling molecule involved in numerous physiological and pathological processes within the human body. This review specifically examines the involvement of NO in age-related diseases, focusing on the cardiovascular, nervous, and immune systems. The discussion delves into the mechanisms of NO signaling in these diseases, emphasizing the post-translational modifications of involved proteins, such as S-nitrosation and nitration. The review also covers the dual nature of NO, highlighting both its protective and harmful effects, determined by concentration, location, and timing. Additionally, potential therapies that modulate NO signaling, including the use of NO donors and nitric oxide synthases (NOSs) inhibitors in the treatment of cardiovascular, neurodegenerative, and oncological diseases, are analyzed. Particular attention is paid to the methods for the determination of NO and its derivatives in the context of illness diagnosis and monitoring. The review underscores the complexity and dual role of NO in maintaining cellular balance and suggests areas for future research in developing new therapeutic strategies.

Species variations in muscle stem cell-mediated immunosuppression on T cells.

Muscle stem cells (MuSCs) are effective in treating inflammatory diseases driven by overactive innate immune responses, such as colitis and acute lung injury, due to their immunomodulatory properties. However, their potential in treating diseases driven by adaptive immune responses is still uncertain. When primed with inflammatory cytokines, MuSCs strongly suppressed T cell activation and proliferation in vitro in co-culture with activated splenocytes or peripheral blood mononuclear cells. Systemic administration of MuSCs from both mice and humans alleviated pathologies in mice with concanavalin A-induced acute liver injury, characterized by hyperactivated T lymphocytes. Importantly, MuSCs showed significant species-specific differences in their immunoregulatory functions. In mouse MuSCs (mMuSCs), deletion or inhibition of inducible nitric oxide synthase (iNOS) reduced their immunosuppressive activity, and absence of iNOS negated their therapeutic effects in liver injury. Conversely, in human MuSCs (hMuSCs), knockdown or inhibition of indoleamine 2,3-dioxygenase (IDO) eliminated their immunosuppressive effects, and loss of IDO function rendered hMuSCs ineffective in treating liver injury in mice. These results reveal significant species-specific differences in the mechanisms by which MuSCs mediate T cell immunosuppression. Mouse MuSCs rely on iNOS, while human MuSCs depend on IDO expression. This highlights the need to consider species-specific responses when evaluating MuSCs' therapeutic potential in immune-related disorders.

Time of day affects MrgD-dependent modulation of cardiomyocyte contractility.

The renin-angiotensin system (RAS) is composed of a series of peptides, receptors, and enzymes that play a pivotal role in maintaining cardiovascular homeostasis. Among the most important players in this system are the angiotensin-II and angiotensin-(1-7) peptides. Our group has recently demonstrated that alamandine (ALA), a peptide with structural and functional similarities to angiotensin-(1-7), interacts with cardiomyocytes, enhancing contractility via the Mas-related G protein-coupled receptor member D (MrgD). It is currently unknown whether this modulation varies along the distinct phases of the day. To address this issue, we assessed the ALA-induced contractility response of cardiomyocytes from mice at four Zeitgeber times (ZTs). At ZT2 (light phase), ALA enhanced cardiomyocyte shortening in an MrgD receptor-dependent manner, which was associated with nitric oxide (NO) production. At ZT14 (dark phase), ALA induced a negative modulation on the cardiomyocyte contraction. β-Alanine, an MrgD agonist, reproduced the time-of-day effects of ALA on myocyte shortening. NG-nitro-l-arginine methyl ester, an NO synthase inhibitor, blocked the increase in fractional shortening induced by ALA at ZT2. No effect of ALA on myocyte shortening was observed at ZT8 and ZT20. Our results show that ALA/MrgD signaling in cardiomyocytes is subject to temporal modulation. This finding has significant implications for pharmacological approaches that combine chronotherapy for cardiac conditions triggered by disruption of circadian rhythms and hormonal signaling.NEW & NOTEWORTHY Alamandine, a member of the renin-angiotensin system, serves critical roles in cardioprotection, including the modulation of cardiomyocyte contractility. Whether this effect varies along the day is unknown. Our results provide evidence that alamandine via receptor MrgD exerts opposing actions on cardiomyocyte shortening, enhancing, or reducing contraction depending on the time of day. These findings may have significant implications for the development and effectiveness of future cardiac therapies.

Immunomodulation of Macrophages in Diabetic Wound Individuals by Structurally Diverse Bioactive Phytochemicals

Diabetes-related ulcers and slow-healing wounds pose a significant health risk to individuals due to their uncertain causes. Mortality rates for diabetes foot ulcers (DFUs) range from 10% after 16 months to 24% after five years. The use of bioactive phytochemicals can play a key role in healing wounds in a predictable time. Recent literature has demonstrated that various natural substances, including flavonoids, saponins, phenolic compounds, and polysaccharides, play key roles at different stages of the wound-healing process through diverse mechanisms. These studies have categorized the compounds according to their characteristics, bioactivities, and modes of action. In this study, we evaluated the role of natural compounds derived from plant sources that have been shown to play a crucial role in immunomodulation. Macrophages are closely involved in immunomodulation within the wound microenvironment and are key players in efferocytosis, inflammation resolution, and tissue regeneration, all of which contribute to successful wound healing. Phytochemicals and their derivatives have shown capabilities in immune regulation, including macrophage migration, nitric oxide synthase inhibition, lymphocyte and T-cell stimulation, cytokine activation, natural killer cell enhancement, and the regulation of NF-κβ, TNF-α, and apoptosis. In this review, we have studied the role of phytochemicals in immunomodulation for the resolution of diabetic wound inflammation.

Single-cell transcriptomics reveal distinctive patterns of fibroblast activation in heart failure with preserved ejection fraction

AbstractInflammation, fibrosis and metabolic stress critically promote heart failure with preserved ejection fraction (HFpEF). Exposure to high-fat diet and nitric oxide synthase inhibitor N[w]-nitro-l-arginine methyl ester (L-NAME) recapitulate features of HFpEF in mice. To identify disease-specific traits during adverse remodeling, we profiled interstitial cells in early murine HFpEF using single-cell RNAseq (scRNAseq). Diastolic dysfunction and perivascular fibrosis were accompanied by an activation of cardiac fibroblast and macrophage subsets. Integration of fibroblasts from HFpEF with two murine models for heart failure with reduced ejection fraction (HFrEF) identified a catalog of conserved fibroblast phenotypes across mouse models. Moreover, HFpEF-specific characteristics included induced metabolic, hypoxic and inflammatory transcription factors and pathways, including enhanced expression of Angiopoietin-like 4 (Angptl4) next to basement membrane compounds, such as collagen IV (Col4a1). Fibroblast activation was further dissected into transcriptional and compositional shifts and thereby highly responsive cell states for each HF model were identified. In contrast to HFrEF, where myofibroblast and matrifibrocyte activation were crucial features, we found that these cell states played a subsidiary role in early HFpEF. These disease-specific fibroblast signatures were corroborated in human myocardial bulk transcriptomes. Furthermore, we identified a potential cross-talk between macrophages and fibroblasts via SPP1 and TNFɑ with estimated fibroblast target genes including Col4a1 and Angptl4. Treatment with recombinant ANGPTL4 ameliorated the murine HFpEF phenotype and diastolic dysfunction by reducing collagen IV deposition from fibroblasts in vivo and in vitro. In line, ANGPTL4, was elevated in plasma samples of HFpEF patients and particularly high levels associated with a preserved global-longitudinal strain. Taken together, our study provides a comprehensive characterization of molecular fibroblast activation patterns in murine HFpEF, as well as the identification of Angiopoietin-like 4 as central mechanistic regulator with protective effects.

The Biological Effects and Mechanisms of Fisetin on Hepatotoxicity, Liver Injury, and Liver Fibrosis: A Systematic Review

Background: Liver disease is a common cause of death worldwide. Objectives: This study aims to investigate the effects and mechanisms of Fisetin on hepatotoxicity, liver injury, and liver fibrosis. Methods: We adhered to the PRISMA 2020 guidelines in this systematic review. Our search used MeSH keywords encompassed Embase, PubMed, Web of Science, Scopus, and Cochrane Library for articles published before March 2, 2024. Relevant data was extracted from the publications, meticulously recorded in a standard form, and subsequently reviewed for outcomes and mechanisms. Results: Fisetin protects hepatocytes from oxidative stress by neutralizing free radicals (O2 −and H2O2), reduces oxidative stress, prevents lipid peroxidation, and increases endogenous antioxidants. It also reduces inflammation via lowering the production of tumor necrosis factor α (TNF-α), interleukins (IL)1α, IL-6, IL-18, IL-1β suppressing nuclear factor kappa B (NF-κB) activation, and cyclooxygenase- 2 (COX-2), inducible nitric oxide synthase (iNOS) inhibition, reducing monocyte chemoattractant protein-1 (MCP-1), plasminogen activator inhibitor 1 (PAI-1), NLR family pyrin domain-containing 3 (NLRP3) inflammasome and interferon-gamma (IFN‐γ). Moreover, it inhibited apoptosis-modulated enzyme activity and detoxification enzymes via modulating the activity of cytochrome P450 and Phase II detoxification enzymes. Fisetin prevented fibrosis by inhibiting the activation of hepatic stellate cells (HSCs), attenuating extracellular matrix (ECM) remodeling-associated genes, and suppressing transforming growth factor-β (TGF-β) signaling pathway and attenuating collagen production. It decreased lipid accumulation and liver function tests. Conclusion: In vivo and in vitro studies indicated that Fisetin can enhance detoxification, attenuate liver injury, and reduce fibrosis, which helps maintain liver health.

Role of l -arginine/nitric oxide/cyclic GMP/K ATP channel signaling pathway and opioid receptors in the antinociceptive effect of rutin in mice.

The l -arginine ( l -Arg)/nitric oxide/cyclic GMP/potassium channel (K ATP ) pathway and opioid receptors are known to play critical roles in pain perception and the antinociceptive effects of various compounds. While there is evidence suggesting that the analgesic effects of rutin may involve nitric oxide modulation, the direct link between rutin and the l -Arg/nitric oxide/cyclic GMP/K ATP pathway in the context of pain modulation requires further investigation. The antinociceptive effect of rutin was studied in male NMRI mice using the formalin test. To investigate the role of the l -Arg/nitric oxide/cyclic GMP/K ATP pathway and opioid receptors, the mice were pretreated intraperitoneally with different substances. These substances included l -Arg (a precursor of nitric oxide), S-nitroso- N -acetylpenicillamine (SNAP, a nitric oxide donor), N(gamma)-nitro- l -arginine methyl ester (L-NAME, an inhibitor of nitric oxide synthase), sildenafil (an inhibitor of phosphodiesterase enzyme), glibenclamide (a K ATP channel blocker), and naloxone (an opioid receptor antagonist). All pretreatments were administered 20 min before the administration of the most effective dose of rutin. Based on our investigation, it was found that rutin exhibited a dose-dependent antinociceptive effect. The administration of SNAP enhanced the analgesic effects of rutin during both the initial and secondary phases. Moreover, L-NAME, naloxone, and glibenclamide reduced the analgesic effects of rutin in both the primary and secondary phases. In conclusion, rutin holds significant value as a flavonoid with analgesic properties, and its analgesic effect is directly mediated through the nitric oxide/cyclic GMP/K ATP channel pathway.