Inhibition of Ferrochelatase Impairs Vascular eNOS/NO and sGC/cGMP Signaling

Ferrochelatase (FECH) is an enzyme necessary for heme synthesis, which is essential for maintaining normal functions of endothelial nitric oxide synthase (eNOS) and soluble guanylyl cyclase (sGC). We tested the hypothesis that inhibition of vascular FECH to attenuate heme synthesis downregulates eNOS and sGC expression, resulting in impaired NO/cGMP-dependent relaxation. To this end, isolated bovine coronary arteries (BCAs) were in vitro incubated without (as controls) or with N-methyl protoporphyrin (NMPP; 10-5-10-7M; a selective FECH antagonist) for 24 and 72 hours respectively. Tissue FECH activity, heme, nitrite/NO and superoxide levels were sequentially measured. Protein expression of FECH, eNOS and sGC was detected by western blot analysis. Vascular responses to various vasoactive agents were evaluated via isometric tension studies. Treatment of BCAs with NMPP initiated a time- and dose-dependent attenuation of FECH activity without changes in its protein expression, followed by significant reduction in the heme level. Moreover, ACh-induced relaxation and ACh-stimulated release of NO were significant reduced, associated with suppression of eNOS protein expression in NMPP-treated groups. Decreased relaxation to NO donor spermine-NONOate reached the statistical significance in BCAs incubated with NMPP for 72 hours, concomitantly with downregulation of sGCbeta1 expression that was independent of heat shock protein 90 (HSP90), nor did it significantly affect BCA relaxation caused by BAY 58-2667 that activates sGC in the heme-deficiency. Neither vascular responses to non-NO/sGC-mediators nor production of superoxide was affected by NMPP-treatment. In conclusion, deletion of vascular heme production via inhibiting FECH elicits downregulation of eNOS and sGC expression, leading to an impaired NO-mediated relaxation in an oxidative stress-independent manner

Sexually Dimorphic Adaptation of Cardiac Function: Roles of Epoxyeicosatrienoic Acid and Peroxisome Proliferator-Activated Receptors

Epoxyeicosatrienoic acids (EETs) are cardioprotective mediators metabolized by soluble epoxide hydrolase (sEH) to form corresponding diols (DHETs). As a sex-susceptible target, sEH is involved in the sexually dimorphic regulation of cardiovascular function. Thus, we hypothesized that the female sex favors EET-mediated potentiation of cardiac function via downregulation of sEH expression, followed by upregulation of peroxisome proliferator-activated receptors (PPARs). Hearts were isolated from male (M) and female (F) wild-type (WT) and sEH-KO mice, and perfused with constant flow at different preloads. Basal coronary flow required to maintain the perfusion pressure at 100 mmHg was significantly greater in females than males, and sEH-KO than WT mice. All hearts displayed a dose-dependent decrease in coronary resistance and increase in cardiac contractility, represented as developed tension in response to increases in preload. These responses were also significantly greater in females than males, and sEH-KO than WT 14,15-EEZE abolished the sex-induced (F vs. M) and transgenic model-dependent (KO vs. WT) differences in the cardiac contractility, confirming an EET-driven response. Compared with M-WT controls, F-WT hearts expressed downregulation of sEH, associated with increased EETs and reduced DHETs, a pattern comparable to that observed in sEH-KO hearts. Coincidentally, F-WT and sEH-KO hearts exhibited increased PPARα expression, but comparable expression of eNOS, PPARβ, and EET synthases. In conclusion, female-specific downregulation of sEH initiates an EET-dependent adaptation of cardiac function, characterized by increased coronary flow via reduction in vascular resistance, and promotion of cardiac contractility, a response that could be further intensified by PPARα

Estrogen-Dependent Epigenetic Regulation of Soluble Epoxide Hydrolase via DNA Methylation

To elucidate molecular mechanisms responsible for the sexually dimorphic phenotype of soluble epoxide hydrolase (sEH) expression, we tested the hypothesis that female-specific down-regulation of sEH expression is driven by estrogen-dependent methylation of the Ephx2 gene. Mesenteric arteries isolated from male, female, ovariectomized female (OV), and OV with estrogen replacement (OVE) mice, as well as the human cell line (HEK293T) were used. Methylation-specific PCR and bisulfite genomic sequencing analysis indicate significant increases in DNA/CG methylation in vessels of female and OVE compared with those of male and OV mice. The same increase in CG methylation was also observed in male vessels incubated with a physiological concentration of 17beta-estradiol (17beta-E2) for 48 hours. All vessels that displayed increases in CG methylation were concomitantly associated with decreases in their Ephx2 mRNA and protein, suggesting a methylation-induced gene silencing. Transient transfection assays indicate that the activity of Ephx2 promoter-coding luciferase was significantly attenuated in HEK293T cells treated with 17beta-E2, which was prevented by additional treatment with an estrogen receptor antagonist (ICI). ChIP analysis indicates significantly reduced binding activities of transcription factors (including SP1, AP-1, and NF-kappaB with their binding elements located in the Ephx2 promoter) in vessels of female mice and human cells treated with 17beta-E2, responses that were prevented by ICI and Decitabine (DNA methyltransferase inhibitor), respectively. In conclusion, estrogen/estrogen receptor-dependent methylation of the promoter of Ephx2 gene silences sEH expression, which is involved in specific transcription factor-directed regulatory pathways

Cytosine-to-Uracil Deamination by SssI DNA Methyltransferase

The prokaryotic DNA(cytosine-5)methyltransferase M.SssI shares the specificity of eukaryotic DNA methyltransferases (CG) and is an important model and experimental tool in the study of eukaryotic DNA methylation. Previously, M.SssI was shown to be able to catalyze deamination of the target cytosine to uracil if the methyl donor S-adenosyl-methionine (SAM) was missing from the reaction. To test whether this side-activity of the enzyme can be used to distinguish between unmethylated and C5-methylated cytosines in CG dinucleotides, we re-investigated, using a sensitive genetic reversion assay, the cytosine deaminase activity of M.SssI. Confirming previous results we showed that M.SssI can deaminate cytosine to uracil in a slow reaction in the absence of SAM and that the rate of this reaction can be increased by the SAM analogue 5’-amino-5’-deoxyadenosine. We could not detect M.SssI-catalyzed deamination of C5-methylcytosine (m5C). We found conditions where the rate of M.SssI mediated C-to-U deamination was at least 100-fold higher than the rate of m5C-to-T conversion. Although this difference in reactivities suggests that the enzyme could be used to identify C5-methylated cytosines in the epigenetically important CG dinucleotides, the rate of M.SssI mediated cytosine deamination is too low to become an enzymatic alternative to the bisulfite reaction. Amino acid replacements in the presumed SAM binding pocket of M.SssI (F17S and G19D) resulted in greatly reduced methyltransferase activity. The G19D variant showed cytosine deaminase activity in E. coli, at physiological SAM concentrations. Interestingly, the C-to-U deaminase activity was also detectable in an E. coli ung+ host proficient in uracil excision repair

Roles of Genetic Predisposition in the Sex Bias of Pulmonary Pathophysiology, as a Function of Estrogens : Sex Matters in the Prevalence of Lung Diseases

In addition to studies focused on estrogen mediation of sex-different regulation of systemic circulations, there is now increasing clinical relevance and research interests in the pulmonary circulation, in terms of sex differences in the morbidity and mortality of lung diseases such as inherent-, allergic- and inflammatory-based events. Thus, female predisposition to pulmonary artery hypertension (PAH) is an inevitable topic. To better understand the nature of sexual differentiation in the pulmonary circulation, and how heritable factors, in vivo- and/or in vitro-altered estrogen circumstances and changes in the live environment work in concert to discern the sex bias, this chapter reviews pulmonary events characterized by sex-different features, concomitant with exploration of how alterations of genetic expression and estrogen metabolisms trigger the female-predominant pathological signaling. We address the following: PAH (Sect.7.2) is characterized as an estrogenic promotion of its incidence (Sect. 7.2.2), as a function of specific germline mutations, and as an estrogen-elicited protection of its prognosis (Sect.7.2.1). More detail is provided to introduce a less recognized gene of Ephx2 that encodes soluble epoxide hydrolase (sEH) to degrade epoxyeicosatrienic acids (EETs). As a susceptible target of estrogen, Ephx2/sEH expression is downregulated by an estrogen-dependent epigenetic mechanism. Increases in pulmonary EETs then evoke a potentiation of PAH generation, but mitigation of its progression, a phenomenon similar to the estrogen-paradox regulation of PAH. Additionally, the female susceptibility to chronic obstructive pulmonary diseases (Sect. 7.3) and asthma (Sect.7.4), but less preference to COVID-19 (Sect. 7.5), and roles of estrogen in their pathogeneses are briefly discussed

Reciprocal Actions of Constrictor Prostanoids and Superoxide in Chronic Hypoxia-Induced Pulmonary Hypertension: Roles of EETs

Epoxyeicosatrienoic acids (EETs) are synthesized from arachidonic acid by CYP/epoxygenase and metabolized by soluble epoxide hydrolase (sEH). Roles of EETs in hypoxia-induced pulmonary hypertension (HPH) remain elusive. The present study aimed to investigate the underlying mechanisms, by which EETs potentiate HPH. Experiments were conducted on sEH knockout (sEH-KO) and wild type (WT) mice after exposure to hypoxia (10% oxygen) for three weeks. In normal/normoxic conditions, WT and sEH-KO mice exhibited comparable pulmonary artery acceleration time (PAAT), ejection time (ET), PAAT/ET ratio, and velocity time integral (VTI), along with similar right ventricular systolic pressure (RVSP). Chronic hypoxia significantly reduced PAAT, ET, and VTI, coincided with an increase in RVSP; these impairments were more severe in sEH-KO than WT mice. Hypoxia elicited downregulation of sEH and upregulation of CYP2C9 accompanied with elevation of CYP-sourced superoxide, leading to enhanced pulmonary EETs in hypoxic mice with significantly higher levels in sEH-KO mice. Isometric tension of isolated pulmonary arteries was recorded. In addition to downregulation of eNOS-induced impairment of vasorelaxation to ACh, HPH mice displayed upregulation of thromboxane A2 (TXA2) receptor, paralleled with enhanced pulmonary vasocontraction to a TXA2 analog (U46619) in an sEH-KO predominant manner. Inhibition of COX-1 or COX-2 significantly prevented the enhancement by approximately 50% in both groups of vessels, and the remaining incremental components were eliminated by scavenging of superoxide with Tiron. In conclusion, hypoxia-driven increases in EETs, intensified COXs/TXA2 signaling, great superoxide sourced from activated CYP2C9, and impaired NO bioavailability work in concert, to potentiate HPH development

Liposomal Nanocarriers of Preassembled Glycocalyx Expeditiously Restore Endothelial Glycocalyx in Endotoxemia

The endothelial glycocalyx (EG) is degraded early during sepsis, and currently available treatments are not effective in promptly restoring it. Here, we created liposomal nanocarriers of preassembled glycocalyx (LNPG) by synthesizing glycosylated syndecan-1 and inserting it into the lipid membrane of unilamellar liposomes. We hypothesized that LNPG would fuse with the endothelial cells where EG is degraded and restore EG in sepsis. We induced endotoxemia in C57BL/6J mice using lipopolysaccharides (LPS) and treated them with LNPG, saline, syndecan-1, or liposomes. LNPG significantly prolonged the survival time of LPS-treated mice compared with the other treatments. Immunostaining of en face mesenteric arteries of LPS-treated mice showed that syndecan-1 was fully restored after LNPG administration. In addition, EG height in microvasculature of mouse cremaster muscle was monitored using sidestream dark field imaging. LNPG restored the perfused boundary region (PBR), which is inversely related to EG dimensions, to the control level after LPS administration. Furthermore, flow-induced dilation in isolated mouse mesenteric arterioles was fully recovered after LNPG treatment in LPS-treated mice. In summary, our findings provide evidence of the therapeutic efficacy of LNPG in the LPS-induced mouse model of sepsis, achieved by expeditiously restoring EG through fusion of LNPG with the endothelial plasma membrane and recovery of endothelial function. Vascular endothelial cells represent the first line of exposure to bacterial endotoxins. Here, we propose a novel therapeutic strategy using liposomes to deliver preassembled glycocalyx to vascular endothelial cell surface and consequently restore endothelial glycocalyx (EG). We tested liposomal nanocarriers of preassembled glycocalyx (LNPG) in vivo and ex vivo to establish for the first time their expeditious therapeutic efficacy in improving survival of lipopolysaccharides (LPS)-treated mice, as achieved by the restoration of EG and recovery of endothelial function

Poster presentation

Quantitative assessment of atrioventricular plane displacement in normal and diastolic heart failure-a cine MRI stud

The Contribution of Chymase-Dependent Formation of ANG II to Cardiac Dysfunction in Metabolic Syndrome of Young Rats: Roles of Fructose and EETs

The roles of ACE-independent ANG II production via chymase and therapeutic potential of epoxyeicosatrienoic acids (EETs) in fructose-induced metabolic syndrome (MetS) in the adolescent population remain elusive. Thus we tested the hypothesis that a high-fructose diet (HFD) in young rats elicits chymase-dependent increases in ANG II production and oxidative stress, responses that are reversible by 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4

Extravascular Blood Augments Myogenic Constriction of Cerebral Arterioles: Implications for Hemorrhage-Induced Vasospasm

BACKGROUND: Subarachnoid hemorrhage is a serious clinical condition that impairs local cerebral blood flow perfusion and consequently initiates neuronal dysfunction. Pressure-sensitive myogenic vasomotor regulation is an important mechanism involved in the regulation of cerebral blood flow. We hypothesized that extravascular hemolyzed blood enhances arteriolar myogenic constriction, which in vivo may contribute to the reduction of local cerebral blood flow after subarachnoid hemorrhage. METHODS AND RESULTS: Arterioles isolated from the middle cerebral artery (MCA arterioles) of mice were cannulated in a perfusion chamber. Arteriolar diameters in response to step increases in intraluminal pressure (20-120 mm Hg) were measured in various experimental conditions. In response to increases in intraluminal pressure, all MCA arterioles exhibited myogenic vasoconstrictions. Compared with controls, the pressure-induced constriction was significantly enhanced in arterioles (in vitro) exposed to extravascular hemolyzed blood or different concentrations of extracellular erythrocyte lysate (1%, 10%, and 20%) for different exposure durations (1-6 hours). The magnitude of enhancement was proportional to the lysate concentration and exposure duration. In in vivo experiments, 10 muL of autologous blood lysate were injected into the mouse subarachnoid space on the surface of the left MCA. Two hours later, MCA arterioles were isolated and left MCA arterioles displayed enhanced myogenic responses compared with the right MCA. The enhanced myogenic response was prevented by scavenge of superoxide in both in vitro and in vivo experiments. CONCLUSIONS: Extravascular hemolyzed blood, perhaps by promoting vascular production of superoxide, augments myogenic constriction of cerebral arterioles, which plays a crucial role in the subarachnoid hemorrhage-induced cerebral ischemia