A Novel Method to Establish a Rat ED Model Using Internal Iliac Artery Ligation Combined with Hyperlipidemia

Objective

To investigate a novel method, namely using bilateral internal iliac artery ligation combined with a high-fat diet (BCH), for establishing a rat model of erectile dysfunction (ED) that, compared to classical approaches, more closely mimics the chronic pathophysiology of human ED after acute ischemic insult.

Materials and Methods

Forty 4-month-old male Sprague Dawley rats were randomly placed into five groups (n = 8 per group): normal control (NC), bilateral internal iliac artery ligation (BIIAL), high-fat diet (HFD), BCH, and mock surgery (MS). All rats were induced for 12 weeks. Copulatory behavior, intracavernosal pressure (ICP), ICP/mean arterial pressure, hematoxylin-eosin staining, Masson''s trichrome staining, serum lipid levels, and endothelial and neuronal nitric oxide synthase immunohistochemical staining of the cavernous smooth muscle and endothelium were assessed. Data were analyzed by SAS 8.0 for Windows.

Results

Serum total cholesterol and triglyceride levels were significantly higher in the HFD and BCH groups than the NC and MS groups. High density lipoprotein levels were significantly lower in the HFD and BCH groups than the NC and MS groups. The ICP values and mount and intromission numbers were significantly lower in the BIIAL, HFD, and BCH groups than in the NC and MS groups. ICP was significantly lower in the BCH group than in the BIIAL and HFD groups. Cavernous smooth muscle and endothelial damage increased in the HFD and BCH groups. Cavernous smooth muscle to collagen ratio, nNOS and eNOS staining decreased significantly in the BIIAL, HFD, and BCH groups compared to the NC and MS groups.

Conclusions

The novel BCH model mimics the chronic pathophysiology of ED in humans and avoids the drawbacks of traditional ED models.     

Vascular endothelial growth factor restores erectile function through modulation of the insulin-like growth factor system and sex hormone receptors in diabetic rat

Previous studies have shown that intracavernous injection of vascular endothelial growth factor (VEGF) restored erectile function in diabetic rats. However, the mechanism of VEGF in diabetes-related erectile dysfunction (ED) has not been fully investigated. We hypothesize that intracavernous injection of VEGF may reverse diabetes-related ED through modulation of the insulin-like growth factor system and sex hormone receptors. To test this hypothesis the erectile function of treated and control rats was analyzed by measurement of intracavernous pressure (ICP) following electrostimulation of the cavernous nerves. Mean ICP was significantly lower in non-treated diabetic rats compared to controls. After VEGF injection, ICP was significantly higher than in non-treated diabetic rats. IGFBP-3 mRNA and protein expression was significantly higher in non-treated diabetic rat crura than controls, while VEGF-treated animals had control levels. ER-beta and PR mRNA and protein expression was significantly lower in non-treated diabetic rat crura. After VEGF injection, ER-beta and PR mRNA and protein expression was similar to control levels. Expression of AR and ER-alpha was the same in all groups. These findings suggest that orthotopic injection of VEGF may improve the functional recovery of diabetes-related ED through modulation of the insulin-like growth factor system and sex hormone receptors. To our knowledge, this is the first study demonstrating that VEGF treatment restores erectile function through restoration of the insulin-like growth factor system and sex hormone receptor genes at the mRNA and protein levels in diabetic rat crura. These results may be important in understanding the pathogenesis of diabetes-related ED and also in providing better strategies for management of this disease.     

Hypoxia Precondition Promotes Adipose-Derived Mesenchymal Stem Cells Based Repair of Diabetic Erectile Dysfunction via Augmenting Angiogenesis and Neuroprotection

The aim of the present study was to examine whether hypoxia preconditioning could improve therapeutic effects of adipose derived mesenchymal stem cells (AMSCs) for diabetes induced erectile dysfunction (DED). AMSCs were pretreated with normoxia (20% O₂, N-AMSCs) or sub-lethal hypoxia (1% O₂, H-AMSCs). The hypoxia exposure up-regulated the expression of several angiogenesis and neuroprotection related cytokines in AMSCs, including vascular endothelial growth factor (VEGF) and its receptor FIK-1, angiotensin (Ang-1), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), stromal derived factor-1 (SDF-1) and its CXC chemokine receptor 4 (CXCR4). DED rats were induced via intraperitoneal injection of streptozotocin (60 mg/kg) and were randomly divided into three groups—Saline group: intracavernous injection with phosphate buffer saline; N-AMSCs group: N-AMSCs injection; H-AMSCs group: H-AMSCs injection. Ten rats without any treatment were used as normal control. Four weeks after injection, the mean arterial pressure (MAP) and intracavernosal pressure (ICP) were measured. The contents of endothelial, smooth muscle, dorsal nerve in cavernoursal tissue were assessed. Compared with N-AMSCs and saline, intracavernosum injection of H-AMSCs significantly raised ICP and ICP/MAP (p<0.05). Immunofluorescent staining analysis demonstrated that improved erectile function by MSCs was significantly associated with increased expression of endothelial markers (CD31 and vWF) (p<0.01) and smooth muscle markers (α-SMA) (p<0.01). Meanwhile, the expression of nNOS was also significantly higher in rats receiving H-AMSCs injection than those receiving N-AMSCs or saline injection. The results suggested that hypoxic preconditioning of MSCs was an effective approach to enhance their therapeutic effect for DED, which may be due to their augmented angiogenesis and neuroprotection.     

Stem Cell Therapy for Diabetic Erectile Dysfunction in Rats: A Meta-Analysis

IntroductionStem cell therapy is a novel method for the treatment of diabetic erectile dysfunction (ED). Many relative animal studies have been done to evaluate the efficacy of this therapy in rats.AimsThis meta-analysis was performed to compare the efficacy of different stem cell therapies, to evaluate the influential factors and to determine the optimal stem cell therapeutic strategy for diabetic ED.MethodsWe searched the studies analyzing the efficacy of stem cell therapy for diabetic ED in rats published before September 30, 2015 in PubMed, Web of Science and EBSCO. A random effects meta-analysis was conducted to assess the outcomes of stem cell therapy. Subgroup analysis was also performed by separating these studies based on their different characteristics. Changes in the ratio of intracavernous pressure (ICP) to mean arterial pressure (MAP) and in the structure of the cavernous body were compared.Results10 studies with 302 rats were enrolled in this meta-analysis. Pooled analysis of these studies showed a beneficial effect of stem cell therapy in improving erectile function of diabetic rats (SMD 4.03, 95% CI = 3.22 to 4.84, P< 0.001). In the stem cell therapy group, both the smooth muscle and endothelium content were much more than those in control group. There was also significant increase in the expression of endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS), the ratio of smooth muscle to collagen, as well as the secretion of vascular endothelial growth factor (VEGF). Besides, apoptotic cells were reduced by stem cell treatment. The subgroup analysis indicated that modified stem cells were more effective than those without modification.ConclusionsOur results confirmed that stem cell therapy could apparently improve the erectile function of diabetic rats. Some specific modification, especially the gene modification with growth factors, could improve the efficacy of stem cell therapy. Stem cell therapy has potential to be an effective therapeutic strategy for diabetic ED.     

Fructose, 1,6-diphosphate mitigates CCl4 suppressed nitric oxide synthase activity in rats

Effect of fructose 1,6-diphosphate (FDP) and carbon tetrachloride (CCl4) were studied individually and in combination on rat endothelial (ET) and smooth muscle cell (SMC) nitric oxide synthase (NOS) activities in vivo, inhibition of ET and SMC NOS activity in CCl4 treated rats was reversed in FDP + CCl4 treated animals. Cellular based NOS activity was significantly increased in FDP treated group of rats when compared to non treated controls. The results suggest a significant increase in NOS in rats treated with a combination of FDP + CCl4 thus overcoming the suppression of NOS exposed to CCl4 alone.     

Alterations of NOS, arginase, and DDAH protein expression in rabbit cavernous tissue after administration of cigarette smoke extract

Cigarette smoking is an independent risk factor for vasculogenic erectile dysfunction (ED). Nitric oxide (NO) has been demonstrated to be the principal mediator of cavernous smooth muscle relaxation and penile erection. Therefore, we examined whether or not enzyme activities and factors involved in the NO generation pathway are affected in rabbit corpus cavernosum after administration of nicotine- and tar-free cigarette smoke extract (CSE). CSE was prepared by bubbling a stream of cigarette smoke into phosphate-buffered saline. CSE was injected subcutaneously into adult male rabbits once a day for 5 wk. In the CSE group, significantly decreased cyclic GMP production as a marker of NO generation was associated with attenuated overall nitric oxide synthase (NOS) activity, enhanced arginase activity, accumulation of endogenous NOS inhibitors such as monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA), and decreased dimethylarginine dimethylaminohydrolase (DDAH) activity as an metabolizing enzyme of endogenous NOS inhibitors. Neuronal NOS (nNOS) and DDAH I protein expression were decreased without altering endothelial NOS expression, while arginase I expression was upregulated. These results suggest that impaired NO production would result from blunted NOS activity, which is possibly brought about by the downregulation of nNOS protein, accumulation of endogenous NOS inhibitors, and enhanced arginase activity together with upregulation of arginase I protein in cavernous tissue. The impaired DDAH activity due to decreased expression of DDAH I protein would result in an accumulation of endogenous NOS inhibitors with CSE. These alterations may be relevant to induction of the erectile dysfunction following CSE.     

Smooth muscle myosin expression, isoform composition, and functional activities in rat corpus cavernosum altered by the streptozotocin-induced type 1 diabetes

Diabetes mellitus (DM) is a quite common chronic disease, and the prevalence of erectile dysfunction (ED) is three times higher in this large population. Although diabetes-related ED has been studied extensively, the actin-myosin contractile apparatus was not examined. The mRNAs encoding smooth muscle myosin (SMM) heavy chains (MHC) and essential light chains (LC(17)) exist as several different alternatively spliced isoforms with distinct contractile properties. Recently, we provided novel data that blebbistatin (BLEB), a specific myosin II inhibitor, potently relaxed corpus cavernosum smooth muscle (CCSM). In this study, we examine whether diabetes alters SMM expression, alternative splicing, and/or functional activities, including sensitivity to BLEB. By using streptozotocin (STZ)-induced 2-mo diabetic rats, functional activities were tested in vivo by intracavernous pressure (ICP) recording during cavernous nerve stimulation and in vitro via organ bath contractility studies. SMM isoform composition was analyzed by competitive RT-PCR and total SMM, myocardin, and embryonic SMM (SMemb) expression by real-time RT-PCR. Results revealed that the blood glucose level of STZ rats was 407.0 vs. 129.5 mg/dl (control). STZ rats exhibited ED confirmed by significantly increased CCSM contractile response to phenylephrine and decreased ICP response. For STZ rats, SM-B, LC(17a) and SM2 isoforms, total SMM, and myocardin expression increased, whereas SM-A, LC(17b), and SM1 isoforms were decreased, with SMemb unchanged. BLEB was significantly more effective in relaxing STZ CCSM both in vitro and in vivo. Thus we demonstrated a novel diabetes-specific effect on alternative splicing of the SMM heavy chain and essential light chain genes to a SMM isoform composition favoring a heightened contractility and ED. A switch to a more contractile phenotype was supported further by total SMM expression increase. Moreover, the change in CCSM phenotype was associated with an increased sensitivity to BLEB, which may serve as a novel pharmacotherapy for ED.     

Antioxidant icariside II combined with insulin restores erectile function in streptozotocin‐induced type 1 diabetic rats

Erectile dysfunction (ED) worsens in patients with diabetes mellitus (DM) despite good control of blood glucose level with insulin. Recent studies imply that diabetic vascular stresses (e.g. oxidative stress) persist in spite of glucose normalization, which is defined as metabolic memory. Studies suggest that the interaction between advanced glycation end products (AGEs) and their receptor (RAGE) mediates the development of metabolic memory. To investigate the effects of the antioxidant icariside II plus insulin on erectile function in streptozotocin (STZ)‐ induced type 1 diabetic rats. Fifty 8‐week‐old Sprague‐Dawley rats were randomly distributed into five groups: normal control, diabetic, insulin‐treated diabetic, icariside II‐treated diabetic, and insulin plus icariside II‐treated diabetic. Diabetes was induced by a single intraperitoneal injection of STZ. Eight weeks after induction of diabetes, icariside II was administered by gastric lavage once a day (5 mg/kg) for 6 weeks; and 2–6 units of intermediate‐acting insulin were given to maintain normal glycemia for 6 weeks. The main outcome measures were the ratio of intracavernous pressure (ICP) to mean arterial pressure (MAP); histology of penile endothelial cells and smooth muscle cells; neural nitric oxide synthase, AGEs and RAGE expression; malondialdehyde concentration; superoxide dismutase activity; and apoptosis index. Diabetic rats demonstrated a significantly lower ICP/MAP ratio, reduced penile endothelial cells, reduced smooth muscle cells, increased AGEs and RAGE, and increased apoptosis. Insulin and icariside II monotherapy partially restored erectile function and histological changes. However, the combination therapy group showed significantly better erectile parameters, cytological components and biochemistry, similar to those in the normal control group. These results suggest that, although insulin can effectively control glycemic levels, it does not completely alter the pathological changes in erectile tissues. Better efficacy could be expected with tight glycemic control plus the antioxidant icariside II. The proposed combination therapy might have the potential to eliminate metabolic memory by down‐regulating the AGEs‐RAGE‐oxidative stress axis.     

Correction of Diabetic Erectile Dysfunction with Adipose Derived Stem Cells Modified with the Vascular Endothelial Growth Factor Gene in a Rodent Diabetic Model

The aim of this study was to determine whether adipose derived stem cells (ADSCs) expressing vascular endothelial growth factor (VEGF) gene can improve endothelial function, recover the impaired VEGF signaling pathway and enhance smooth muscle contents in a rat diabetic erectile dysfunction (DED) model. DED rats were induced via intraperitoneal injection of streptozotocin (40 mg/kg), and then screened by apomorphine (100 µg/kg). Five groups were used (n = 12/group)–Group 1 (G1): intracavernous injection of lentivirus-VEGF; G2: ADSCs injection; G3: VEGF-expressing ADSCs injection; G4: Phosphate buffered saline injection; G1–G4 were DED rats; G5: normal rats. The mean arterial pressure (MAP) and intracavernosal pressure (ICP) were measured at days 7 and 28 after the injections. The components of the VEGF system, endothelial, smooth muscle, pericytes markers in cavernoursal tissue were assessed. On day 28 after injection, the group with intracavernosum injection of ADSCs expressing VEGF displayed more efficiently and significantly raised ICP and ICP/MAP (p<0.01) than those with ADSCs or lentivirus-VEGF injection. Western blot and immunofluorescent analysis demonstrated that improved erectile function by ADSCs-VEGF was associated with increased expression of endothelial markers (VEGF, VEGF R1, VEGF R2, eNOS, CD31 and vWF), smooth muscle markers (a-actin and smoothelin), and pericyte markers (CD146 and NG2). ADSCs expressing VEGF produced a therapeutic effect and restored erectile function in diabetic rats by enhancing VEGF-stimulated endothelial function and increasing the contents of smooth muscle and pericytes.     

Knockdown of connective tissue growth factor by plasmid-based short hairpin RNA prevented pulmonary vascular remodeling in cigarette smoke-exposed rats

Cigarette smoking may contribute to pulmonary hypertension in chronic obstructive pulmonary disease by resulting in pulmonary vascular remodeling that involves pulmonary artery smooth muscle cell proliferation. Connective tissue growth factor (CTGF) is a cysteine-rich peptide implicated in several biological processes such as cell proliferation, survival, and migration. This study investigated the potential role of CTGF in pulmonary vascular remodeling. We constructed a plasmid-based short hairpin RNA (shRNA) to knock down the expression of CTGF in primary cultured rat pulmonary artery smooth muscle cells (rPASMCs) and in rat lung vessels. Rat PASMCs were challenged with cigarette smoke extract (CSE). Rats were exposed to cigarette smoke for 3 months in the absence or in the presence of plasmid-based short hairpin RNA against CTGF which was administrated by tail vein injection. CTGFshRNA significantly prevented CTGF and cyclin D1 expression, arrested cell cycle at G0/G1 phase and suppressed cell proliferation in rPASMCs exposed to CSE. CTGFshRNA administration ameliorated pulmonary vascular remodeling, inhibited cigarette smoke-induced CTGF elevation and reversed the cyclin D1 increase in pulmonary vessels in rats. Collectively, our data demonstrated that plasmid-based shRNA against CTGF attenuated pulmonary vascular remodeling in cigarette smoke-exposed rats.     

Lack of central nitric oxide triggers erectile dysfunction in diabetes

Erectile dysfunction is a serious and common complication of diabetes mellitus. The proposed mechanisms for erectile dysfunction in diabetes include central and autonomic neuropathy, endothelial dysfunction, and smooth muscle dysfunction. The paraventricular nucleus (PVN) of the hypothalamus is known to be involved in centrally mediated penile erection. This study was designed to examine the role of nitric oxide (NO) within the central nervous system component of the behavioral responses including erection in diabetic rats. N-methyl-D-aspartic acid (NMDA)-induced erection, yawning, and stretch through the PVN can be blocked by prior administration of NO synthase (NOS) blocker, L-NMMA, in freely moving, conscious male normal rats. Four weeks after streptozotocin (STZ) and vehicle injections, NMDA-induced erection, yawning, and stretch responses through the PVN are significantly blunted in diabetic rats compared with control rats. Examination of neuronal NOS (nNOS) protein by Western blot analysis indicated a reduced amount of nNOS protein in the PVN of rats with diabetes compared with control rats. Furthermore, restoring nNOS within the PVN by gene transfer using adenoviral transfection significantly restored the erectile and yawning responses to NMDA in diabetic rats. These data demonstrate that a blunted NO mechanism within the PVN may contribute to NMDA-induced erectile dysfunction observed in diabetes mellitus.     

Nitric oxide produced via neuronal NOS may impair vasodilatation in septic rat skeletal muscle

Impaired vascular responsiveness in sepsis may lead to maldistribution of blood flow in organs. We hypothesized that increased production of nitric oxide (NO) via inducible nitric oxide synthase (iNOS) mediates the impaired dilation to ACh in sepsis. Using a 24-h cecal ligation and perforation (CLP) model of sepsis, we measured changes in arteriolar diameter and in red blood cell velocity (V(RBC)) in a capillary fed by the arteriole, following application of ACh to terminal arterioles of rat hindlimb muscle. Sepsis attenuated both ACh-stimulated dilation and V(RBC) increase. In control rats, arteriolar pretreatment with the NO donors S-nitroso-N-acetylpenicillamine or sodium nitroprusside reduced diameter and V(RBC) responses to a level that mimicked sepsis. In septic rats, arteriolar pretreatment with the "selective" iNOS blockers aminoguanidine (AG) or S-methylisothiourea sulfate (SMT) restored the responses to the control level. The putative neuronal NOS (nNOS) inhibitor 7-nitroindazole also restored the response toward control. At 24-h post-CLP, muscles showed no reduction of endothelial NOS (eNOS), elevation of nNOS, and, surprisingly, no induction of iNOS protein; calcium-dependent constitutive NOS (eNOS+nNOS) enzyme activity was increased whereas calcium-independent iNOS activity was negligible. We conclude that 1) AG and SMT inhibit nNOS activity in septic skeletal muscle, 2) NO could impair vasodilative responses in control and septic rats, and 3) the source of increased endogenous NO in septic muscle is likely upregulated nNOS rather than iNOS. Thus agents released from the blood vessel milieu (e.g., NO produced by skeletal muscle nNOS) could affect vascular responsiveness.     

Protection from cigarette smoke‐induced vascular injury by recombinant human relaxin‐2 (serelaxin)

Smoking is regarded as a major risk factor for the development of cardiovascular diseases (CVD). This study investigates whether serelaxin (RLX, recombinant human relaxin‐2) endowed with promising therapeutic properties in CVD, can be credited of a protective effect against cigarette smoke (CS)‐induced vascular damage and dysfunction. Guinea pigs exposed daily to CS for 8 weeks were treated with vehicle or RLX, delivered by osmotic pumps at daily doses of 1 or 10 μg. Controls were non‐smoking animals. Other studies were performed on primary guinea pig aortic endothelial (GPAE) cells, challenged with CS extracts (CSE) in the absence and presence of 100 ng/ml (17 nmol/l) RLX. In aortic specimens from CS‐exposed guinea pigs, both the contractile and the relaxant responses to phenylephrine and acetylcholine, respectively, were significantly reduced in amplitude and delayed, in keeping with the observed adverse remodelling of the aortic wall, endothelial injury and endothelial nitric oxide synthase (eNOS) down‐regulation. RLX at both doses maintained the aortic contractile and relaxant responses to a control‐like pattern and counteracted aortic wall remodelling and endothelial derangement. The experiments with GPAE cells showed that CSE significantly decreased cell viability and eNOS expression and promoted apoptosis by sparkling oxygen free radical‐related cytotoxicity, while RLX counterbalanced the adverse effects of CSE. These findings demonstrate that RLX is capable of counteracting CS‐mediated vascular damage and dysfunction by reducing oxidative stress, thus adding a tile to the growing mosaic of the beneficial effects of RLX in CVD.     

Primary cultures of rat aortic endothelial and smooth muscle cells: I. An in vitro model to study xenobiotic-induced vascular cytotoxicity

Summary Primary cultures of rat vascular endothelial and smooth muscle cells were developed as models to study xenobiotic-induced cytotoxicity. Endothelial and smooth muscle cells were isolated by enzymatic digestion and mechanical dissociation of rat thoracic aortae. Optimal cell growth and minimal fibroblast contamination in cultures of both cell types were obtained in Medium 199 supplemented with 10% fetal bovine serum. Cultured cells were characterized by distinctive morphologic features and growth patterns. Intercellular endothelial cell junctions were selectively stained with silver nitrate. Endothelial cells also exhibited a nonthrombogenic surface, as reflected by platelet-binding studies. Confluent cultures of smooth muscle cells, but not endothelial cells, contracted in response to norepinephrine (10 μM). Cultures of both cell types were exposed to acrolein (2, 5 or 50 ppm), an environmental pollutant, for 4 24 h. Morphologic damage, lactate dehydrogenase release, and cellular thiol content were used as indices of cytotoxicity. Acrolein-induced enzyme leakage and morpholgic alterations were dose- and time-dependent and more pronounced in cultures of smooth muscle cells than in endothelial cells. The total thiol content of endothelial cells exposed to acrolein (50 ppm) for 24 h was not significantly different from that of respective controls. In contrast, the content of treated smooth muscle cells was higher than that of controls. These observations show that primary cultures of vascular cells provide a useful model to evaluate xenobiotic-induced cytotoxicity. The information obtained using a cell culture system may be complemented by the use of other in vivo and in vitro models to determine the mechanisms by which xenobiotics cause vascular cell injury.     

Cerebral blood flow autoregulation in early experimental S. pneumoniae meningitis.

We studied cerebral blood flow (CBF) autoregulation and intracranial pressure (ICP) during normo- and hyperventilation in a rat model of Streptococcus pneumoniae meningitis. Meningitis was induced by intracisternal injection of S. pneumoniae. Mean arterial blood pressure (MAP), ICP, cerebral perfusion pressure (CPP, defined as MAP - ICP), and laser-Doppler CBF were measured in anesthetized infected rats (n = 30) and saline-inoculated controls (n = 30). CPP was either incrementally reduced by controlled hemorrhage or increased by intravenous norepinephrine infusion. Twelve hours postinoculation, rats were studied solely during normocapnia, whereas rats studied after 24 h were exposed to either normocapnia or to acute hypocapnia. In infected rats compared with control rats, ICP was unchanged at 12 h but increased at 24 h postinoculation (not significant and P < 0.01, respectively); hypocapnia did not lower ICP compared with normocapnia. Twelve hours postinoculation, CBF autoregulation was lost in all infected rats but preserved in all control rats (P < 0.01). Twenty-four hours after inoculation, 10% of infected rats had preserved CBF autoregulation during normocapnia compared with 80% of control rats (P < 0.01). In contrast, 60% of the infected rats and 100% of the control rats showed an intact CBF autoregulation during hypocapnia (P < 0.05 for the comparison of infected rats at normocapnia vs. hypocapnia). In conclusion, CBF autoregulation is lost both at 12 and at 24 h after intracisternal inoculation of S. pneumoniae in rats. Impairment of CBF autoregulation precedes the increase in ICP, and acute hypocapnia may restore autoregulation without changing the ICP.     

Alterations in Skeletal Muscle Cell Homeostasis in a Mouse Model of Cigarette Smoke Exposure

Background

Skeletal muscle dysfunction is common in chronic obstructive pulmonary disease (COPD), a disease mainly caused by chronic cigarette use. An important proportion of patients with COPD have decreased muscle mass, suggesting that chronic cigarette smoke exposure may interfere with skeletal muscle cellular equilibrium. Therefore, the main objective of this study was to investigate the kinetic of the effects that cigarette smoke exposure has on skeletal muscle cell signaling involved in protein homeostasis and to assess the reversibility of these effects.

Methods

A mouse model of cigarette smoke exposure was used to assess skeletal muscle changes. BALB/c mice were exposed to cigarette smoke or room air for 8 weeks, 24 weeks or 24 weeks followed by 60 days of cessation. The gastrocnemius and soleus muscles were collected and the activation state of key mediators involved in protein synthesis and degradation was assessed.

Results

Gastrocnemius and soleus were smaller in mice exposed to cigarette smoke for 8 and 24 weeks compared to room air exposed animals. Pro-degradation proteins were induced at the mRNA level after 8 and 24 weeks. Twenty-four weeks of cigarette smoke exposure induced pro-degradation proteins and reduced Akt phosphorylation and glycogen synthase kinase-3β quantity. A 60-day smoking cessation period reversed the cell signaling alterations induced by cigarette smoke exposure.

Conclusions

Repeated cigarette smoke exposure induces reversible muscle signaling alterations that are dependent on the duration of the cigarette smoke exposure. These results highlights a beneficial aspect associated with smoking cessation.     

Effects of repeated antigen exposure on endothelin-1-induced bronchial smooth muscle contraction and activation of RhoA in sensitized rats

Changes in endothelin-1 (ET-1)-induced contraction and activation of RhoA in bronchial smooth muscle of repeatedly antigen-challenged rats, which exhibit marked airway hyperresponsiveness (AHR), were examined. The ET-1-induced contraction of bronchial smooth muscle was significantly enhanced in the repeatedly antigen-challenged group. In normal control animals, ET-1 induced time- and concentration-dependent translocation of RhoA to the plasma membrane, indicating activation of RhoA by ET-1 in rat bronchial smooth muscle. The level of ET-1-induced RhoA translocation was increased much more markedly in the AHR group than in the control animals. It is suggested that the augmented activation of RhoA observed in the hyperresponsive bronchial smooth muscle might be responsible for the enhanced ET-1-induced contraction of bronchial smooth muscle in AHR rats.     

Chronic O2 exposure in the newborn rat results in decreased pulmonary arterial nitric oxide release and altered smooth muscle response to isoprostane.

Chronic oxygen exposure in the newborn rat results in lung isoprostane formation, which may contribute to the pulmonary hypertension evident in this animal model. The purpose of this study was to investigate the pulmonary arterial smooth muscle responses to 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2a)) in newborn rats exposed to 60% O2 for 14 days. Because, in the adult rat, 8-iso-PGF(2alpha) may have a relaxant effect, mediated by nitric oxide (NO), we also sought to evaluate the pulmonary arterial NO synthase (NOS) protein content and NO release in the newborn exposed to chronic hyperoxia. Compared with air-exposed control animals, 8-iso-PGF(2a) induced a significantly greater force (P < 0.01) and reduced (P < 0.01) relaxation of precontracted pulmonary arteries in the 60% O2-treated animals. These changes were reproduced in control pulmonary arteries by NOS blockade by using NG-nitro-L-arginine methyl ester. Pulmonary arterial endothelial NOS was unaltered, but the inducible NOS protein content was significantly decreased (P < 0.01) in the experimental group. Pulmonary (P < 0.05) and aortic (P < 0.01) tissue ex vivo NO accumulation was significantly reduced in the 60% O2-treated animals. We speculate that impaired pulmonary vascular tissue NO metabolism after chronic O2 exposure potentiates 8-iso-PGF(2alpha)-induced vasoconstriction in the newborn rat, thus contributing to pulmonary hypertension.     

Oltipraz protects the passive smoke induced changes in renal glyoxalase system of rats

The present study was performed to investigate the effect of oltipraz on passive smoke-induced alteration in renal glyoxalase system of rats. Adult Sprague-Dawley rats were exposed daily to passive cigarette smoke in a whole-body exposure chamber 6 h per day for 2, 4 and 12 weeks. The animals being sacrificed after 2 and 12 weeks were maintained on control diet, powdered 4% Teklad rat chow (Harlan Teklad, Madison, WI, USA). The 4 weeks group was divided into three subgroups, one receiving control diet, other two receiving control diet supplemented with two doses of oltipraz (either 167 or 500 ppm), starting 1 week prior to initiation of smoke exposure until the end of the experiment. The activity of glyoxalase I was higher in animals exposed for 4 and 12 weeks of passive smoke than those exposed for 2 weeks. There was no significant difference between 4 and 12 weeks. Glyoxalase II activity was lower in animals exposed to passive smoke for 4 weeks than those exposed for 2 weeks. However, the activity approached the basal level after 12 weeks of exposure. Furthermore, oltipraz treatment maintained the activity of both glyoxalase closer to the basal levels.