MicroRNA-17-5p, a novel endothelial cell modulator, controls vascular re-endothelialization and neointimal lesion formation

Background The functions of miR-17-5p in tumorigenesis have been explored. However, their functionalities in arterial endothelial cells (ECs) have not been investigated. Besides, the issue of vascular remodelling is barely addressed. Objectives The study aimed to determine the effect of overexpression or inhibition of miR-17-5p on arterial endothelial cells’ (ECs) function and vascular remodelling in vitro and the rat carotid arteries model. Methods Quantitative RT-PCR analysis was performed to examine the expression of miR-17-5p. Then, gain-of-function and loss-of-function approaches were employed to investigate the functional roles of miR-17-5p in cultured human coronary artery endothelial cells (HCAECs); further, TargetScan software analysis and luciferase reporter activity assay were performed to investigate the potential mechanism. Lastly, the results of the cell segment were verified in a rat carotid artery balloon injury model by Western blot analysis, measurement of the vascular cGMP level and plasma 8-iso-prostaglandin F2 (8-iso-PGF2) testing. Moreover, morphometric analysis was implemented to detect the re-endothelialization and neointimal formation in rat carotid artery after balloon injury. Results This study firstly found that miR-17-5p expression was upregulated in the injured vascular walls and highly expressive in ECs; overexpression of miR-17-5p inhibited HCAECs’ proliferation and migration, whereas miR-17-5p knockdown strengthened its proliferative and migratory roles, influenced inflammatory response, through regulating VEGRA and VEGFR2. It was found that miR-17-5p bind to VEGFA and VEGFR2 at the 3′UTR. Next, downregulation of miR-17-5p promotes re-endothelialization, and attenuates neointimal formation as measured by the I/M ratio (0.63±0.05 vs 1.45±0.06, antagomiR-17-5p vs. Lenti-NC, p < 0.05). In addition, the functional recovery of the endothelium was also accelerated by miR-17-5p knockdown. Conclusion Our study suggests that miR-17-5p is a feasible strategy for the selective modulation of endothelialization and vascular remodelling through regulating VEGFA and VEGFR2.

Assessment of cGMP level in medium during in vitro growth period of murine preantral follicles with and without supplementation of C-type natriuretic peptide

Summary To enhance the developmental competency of murine ovarian follicles cultured in vitro, C-type natriuretic peptide (CNP) was supplemented in the culture system. Although the mechanism is not fully elucidated, it was reported that the effect of CNP supplementation was mediated by increased cyclic guanosine monophosphate (cGMP). In the present study, cGMP levels in media for murine preantral follicle culture were compared both between a control group without CNP supplementation and an experimental group with CNP supplementation and between days in each group. In addition, follicle growth patterns and oocyte maturity were assessed and compared between the two groups. Results demonstrated that along with in vitro culture, cGMP levels increased (P < 0.05) both in the control group and the experimental group, whereas cGMP levels were not significantly different between the two groups on the same day of in vitro culture (P > 0.05). The oocyte’s maturity was superior in the experimental group compared with the control group (P < 0.05). As ovarian follicles grew three-dimensionally in the experimental group but were flattened in the control group, CNP might improve oocyte maturity through maintaining the three-dimensional architecture of the ovarian follicle because of increased transzonal projections (TZP) and functional gap junctions between oocyte and surrounding granulosa cells.

An engineered membrane-bound guanylyl cyclase with light-switchable activity

Background Microbial rhodopsins vary in their chemical properties, from light sensitive ion transport to different enzymatic activities. Recently, a novel family of two-component Cyclase (rhod)opsins (2c-Cyclop) from the green algae Chlamydomonas reinhardtii and Volvox carteri was characterized, revealing a light-inhibited guanylyl cyclase (GC) activity. More genes similar to 2c-Cyclop exist in algal genomes, but their molecular and physiological functions remained uncharacterized. Results Chlamyopsin-5 (Cop5) from C. reinhardtii is related to Cr2c-Cyclop1 (Cop6) and can be expressed in Xenopus laevis oocytes, but shows no GC activity. Here, we exchanged parts of Cop5 with the corresponding ones of Cr2c-Cyclop1. When exchanging the opsin part of Cr2c-Cyclop1 with that of Cop5, we obtained a bi-stable guanylyl cyclase (switch-Cyclop1) whose activity can be switched by short light flashes. The GC activity of switch-Cyclop1 is increased for hours by a short 380 nm illumination and switched off (20-fold decreased) by blue or green light. switch-Cyclop1 is very light-sensitive and can half-maximally be activated by ~ 150 photons/nm2 of 380 nm (~ 73 J/m2) or inhibited by ~ 40 photons/nm2 of 473 nm (~ 18 J/m2). Conclusions This engineered guanylyl cyclase is the first light-switchable enzyme for cGMP level regulation. Light-regulated cGMP production with high light-sensitivity is a promising technique for the non-invasive investigation of the effects of cGMP signaling in many different tissues.

Inhibition of Phosphodiesterase 5 Promotes the Aromatase-Mediated Estrogen Biosynthesis in Osteoblastic Cells by Activation of cGMP/PKG/SHP2 Pathway

Mechanical stimulation induces bone growth and remodeling by the secondary messenger, cyclic guanosine 3’, 5’-monophosphate (cGMP), in osteoblasts. However, the role of cGMP in the regulation of estrogen biosynthesis, whose deficiency is a major cause of osteoporosis, remains unclear. Here, we found that the prenylated flavonoids, 3-O-methoxymethyl-7-O-benzylicaritin (13), 7-O-benzylicaritin (14), and 4'-O-methyl-8-isopentylkaempferol (15), which were synthesized using icariin analogs, promoted estrogen biosynthesis in osteoblastic UMR106 cells, with calculated EC50 values of 1.53, 3.45, and 10.57 µM, respectively. 14 and 15 increased the expression level of the bone specific promoter I.4-driven aromatase, the only enzyme that catalyzes estrogen formation by using androgens as substrates, in osteoblastic cells. 14 inhibited phosphodiesterase 5 (PDE5), stimulated intracellular cGMP level and promoted osteoblast cell differentiation. Inhibition of cGMP dependent-protein kinase G (PKG) abolished the stimulatory effect of 14 on estrogen biosynthesis and osteoblast cell differentiation. Further, PKG activation by 14 stimulated the activity of SHP2 (Src homology 2 domain-containing tyrosine phosphatase 2), thereby activating Src and ERK (extracellular signal-regulated kinase) signaling and increasing ERK-dependent aromatase expression in osteoblasts. Our findings reveal a previously unknown role of cGMP in the regulation of estrogen biosynthesis in the bone. These results support the further development of 14 as a PKG-activating drug to mimic the anabolic effects of mechanical stimulation of bone in the treatment of osteoporosis.

Neuroprotective Effects of Sildenafil on Traumatic Brain Injury in an Experimental Rat Model

Objective Not only primary injuries, secondary injuries such as posttraumatic biochemical cascades, ischemia, and hypoxia also affect the morbidity and mortality of traumatic brain injury (TBI). Sildenafil released the vasodilatation by relaxing the smooth muscle of the systemic artery and vein. Also, the effects of sildenafil are evidenced in multiple sclerosis, Alzheimer's disease, and memory loss as a part of experimental studies. Sildenafil decreases oxidative stress by increasing the cGMP level. We aimed to examine the protective effects of sildenafil on TBI with histopathological and biochemical parameters. Method 21 Sprague–Dawley rats were separated into three groups (n = 7). “The weight drop injury model,” which was described by Marmou, was used for the head injury. Group 1: nontraumatic sham group, Group 2: nontreated TBI group, Group 3: sildenafil (100 mg/kg) treated TBI group. The whole brain and serum were collected for histopathological and biochemical study. The histopathological sections were examined under a light microscope. Results On comparison of total antioxidant status (TAS), total oxidant status (TOS), nitric oxide (NO), and plasma nitrite/nitrate (PNOx) between groups, NO level was significantly high in group 3 (p = 0.013). Even though the TAS level was significantly high in group 3 (p = 0.02), there were no significant differences in TOS level in groups (p = 0.225). Disappearing Nissle granules occurred in a pyknotic situation in the cell nucleus, and acidophilic staining in neuron cells, which describe the neuron degeneration observed in the trauma group. The neuron degeneration markers were not seen in the sildenafil-treated trauma group. Conclusion Our study has shown that sildenafil decreases the oxygen radicals and affects the recovery of experimental TBI in rats.

Orlistat reverses intratesticular lactate transport decline and infertility in male obese rats

Obesity and its accompanying complications predispose to abnormal testicular glucose metabolism, penile erectile dysfunction and subfertility. This study examined the potentials of orlistat in attenuating erectile dysfunction and fertility decline in high-fat diet (HFD)-induced obesity in male rats. Eighteen adult male Sprague–Dawley rats whose weights were between 250 and 300 g were divided into three groups (n = 6/group) namely: normal control (NC), HFD and HFD + orlistat (10 mg/kg body weight/day co-administered for 12 weeks) (HFD+O). During the 11th and 12th week, mating behaviour and fertility parameters were evaluated, and parameters of glucose metabolism were assessed at the end of the 12th week. Orlistat increased testicular mRNA levels of glucose transporters (Glut1 and Glut3), monocarboxylate transporters (Mct2 and Mct4) and lactate dehydrogenase type C (Ldhc), decreased intratesticular lactate and glucose levels, and LDH activity in obese rats. Furthermore, orlistat increased superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and glutathione reductase (GR) activities, and total antioxidant capacity (TAC), but decreased malondialdehyde level in the penis of obese rats. Similarly, orlistat improved penile cGMP level, sexual behaviour and fertility outcome in obese rats. Penile cGMP level correlated positively with total mounts and intromissions but correlated negatively with mount/intromission ratio. Orlistat improves fertility potential in obese state by targeting testicular lactate metabolism, penile oxidative stress and sexual behaviour in rats. Therefore, orlistat shows a promising protective effect and may preserve the fertility potential of obese men.

Abstract 12567: Circulating Factors Induce Cardiomyopathy After Burn Injury

Introduction: Our previous results in vivo indicated PDE5-cGMP-PKG was involved in burn-induced heart dysfunction and PDE5A inhibitor restored the dysfunction. It’s unknown if circulating factors after burn would injure cardiomyocytes. Hypothesis: Circulating factors released after burn induce cardiomyopathy. Methods: Human cardiomyocytes (AC16) were treated with sham-serum, burn-serum (24 hpb-serum) and burn/sildenafil-serum (24 hpb/SIL). We performed cut-edged biochemistry technologies and Illumina RNA sequencing (RNA-seq) in this study. GraphPad Prism 8.4.2 was used for statistics. Results: We found a significant decrease of cGMP level and an increase of cTN1 in 24 hpb-serum group. Treatment with the PDE5A inhibitor Sildenafil completely reversed this change similar to our in vivo work. To understand what bioactive molecules would be involved in the alterations by burn injury, human cardiomyocytes (Ac16) were employed to test the cardiomyocyte response to burn-induced circulating factors. We observed that 24 hpb-serum significantly 1) decreased cell viability and cell proliferation; as well as 2) increased cell cytotoxicity, cell apoptosis and cell ROS production. We also found 24 hpb-serum resulted in cell mitochondrial dysfunction by decreasing ATP production and mitochondrial membrane integrity/potential and increasing mitochondrial ROS. Seahorse and O2K approaches confirmed 24 hpb-serum-induced cardiomyocyte mitochondrial dysfunction as evidenced by decreases of mitochondrial basal respiration, proton leak, ATP production, and maximal respiration. 24 hpb/SIL serum rescued 24 hpb serum-induced Ac 16 cell response, at least partially. Advanced bioinformatic analyses identified 1415 upregulated genes and 1091 downregulated genes in 24 hpb-serum group and 776 upregulated genes and 113 downregulated genes restored in 24 hpb/SIL-serum group. We also analyzed and validated the differentially expressed genes. Conclusions: Our study not only confirmed burn induced heart dysfunction, but also provided evidence for understanding the pathogenic mechanism of circulating factors released after burn injury and preliminary genomic evidence for the mechanism for cardiomyopathy after burn injury.

Homogeneous single-label cGMP detection platform for the functional study of nitric oxide-sensitive (soluble) guanylyl cyclases and cGMP-specific phosphodiesterases

Cardiovascular diseases are the number one death worldwide. Nitric oxide (NO)—NO-sensitive (soluble) guanylyl cyclase (sGC)—cyclic guanosine monophosphate (cGMP) pathway regulates diverse set of important physiological functions, including maintenance of cardiovascular homeostasis. Resting and activated sGC enzyme converts guanosine triphosphate to an important second messenger cGMP. In addition to traditional NO generators, a number of sGC activators and stimulators are currently in clinical trials aiming to support or increase sGC activity in various pathological conditions. cGMP-specific phosphodiesterases (PDEs), which degrade cGMP to guanosine monophosphate, play key role in controlling the cGMP level and the strength or length of the cGMP-dependent cellular signaling. Thus, PDE inhibitors also have clear clinical applications. Here, we introduce a homogeneous quenching resonance energy transfer (QRET) for cGMP to monitor both sGC and PDE activities using high throughput screening adoptable method. We demonstrate that using cGMP-specific antibody, sGC or PDE activity and the effect of small molecules modulating their function can be studied with sub-picomole cGMP sensitivity. The results further indicate that the method is suitable for monitoring enzyme reactions also in complex biological cellular homogenates and mixture.

Sildenafil Recovers Burn-Induced Cardiomyopathy

Background: Severe burn injury initiates a feedback cycle of inflammation, fibrosis, oxidative stress and cardiac mitochondrial damage via the PDE5A-cGMP-PKG pathway. Aim: To test if the PDE5A-cGMP-PKG pathway may contribute to burn-induced heart dysfunction. Methods: Sprague–Dawley rats were divided four groups: sham; sham/sildenafil; 24 h post burn (60% total body surface area scald burn, harvested at 24 h post burn); and 24 h post burn/sildenafil. We monitored heart function and oxidative adducts, as well as cardiac inflammatory, cardiac fibrosis and cardiac remodeling responses in vivo. Results: Sildenafil inhibited the burn-induced PDE5A mRNA level and increased the cGMP level and PKG activity, leading to the normalization of PKG down-regulated genes (IRAG, PLB, RGS2, RhoA and MYTP), a decreased ROS level (H2O2), decreased oxidatively modified adducts (malonyldialdehyde [MDA], carbonyls), attenuated fibrogenesis as well as fibrosis gene expression (ANP, BNP, COL1A2, COL3A2, αSMA and αsk-Actin), and reduced inflammation and related gene expression (RELA, IL-18 and TGF-β) after the burn. Additionally, sildenafil treatment preserved left ventricular heart function (CO, EF, SV, LVvol at systolic, LVPW at diastolic and FS) and recovered the oxidant/antioxidant balance (total antioxidant, total SOD activity and Cu,ZnSOD activity). Conclusions: The PDE5A-cGMP-PKG pathway mediates burn-induced heart dysfunction. Sildenafil treatment recovers burn-induced cardiac dysfunction.

Muscle fiber type differences in nitrate and nitrite storage and nitric oxide signaling in rats

Recent studies have emphasized the importance of the nitric oxide synthase (NOS)-independent, nitrate (NO3−) → nitrite (NO2−) → nitric oxide (NO) pathway in skeletal muscle. In particular, it has been hypothesized that this pathway is especially active in type II, or fast-twitch, muscle fibers, necessitating greater NO3− and NO2− storage. We therefore measured NO3− and NO2− concentrations in the predominantly fast-twitch vastus lateralis and predominantly slow-twitch soleus muscles of rats. Contrary to the above hypothesis, we found that NO3− and NO2− concentrations were 3.4-fold and 1.8-fold higher, respectively, in the soleus. On the other hand, NO signaling (i.e., cyclic guanosine monophosphate (cGMP) level) was comparable in the two muscles. Although the physiological significance of these observations remains to be determined, we speculate that NO production via the NO3− → NO2− → NO pathway is normally higher in slow-twitch muscles, thus helping compensate for their inherently lower NOS activity.