Dysregulation of PTEN in Cardiopulmonary Vascular Remodeling Induced by Pulmonary Hypertension

Pulmonary hypertension (PH) is a disorder of lung vasculature characterized by arterial narrowing. Phosphatase-and-tensin homolog on chromosome 10 (PTEN), associated in the progression of multiple cancers, is implicated in arterial remodeling. However, the involvement of PTEN in PH remains unclear. The objective of the present study was to determine the role of PTEN in pulmonary vascular remodeling using established models of PH. The study used rat models of PH, induced by monocrotaline (MCT) administration (60 mg/kg) or continuous hypoxic exposure (10% oxygen) for 3 weeks. Pulmonary artery smooth muscle cells (SMCs) were used for in vitro confirmation. Development of PH was verified by hemodynamic, morphological and histopathology analyses. PTEN and key downstream proteins in pulmonary and cardiac tissues were analyzed by western blotting and RT-PCR. PTEN was significantly decreased (MCT, 53%; Hypoxia, 40%), pAkt was significantly increased (MCT, 42%; Hypoxia, 55%) in tissues of rats with PH. Similar results were observed in SMCs exposed to hypoxia (1% oxygen) for 48 h. Ubiquitination assay showed that PTEN degradation occurs via proteasomal degradation pathway. Western blotting demonstrated a significant downregulation of cell-cycle regulatory proteins p53 and p27, and upregulation of cyclin-D1 in the lungs of both models. The results showed that PTEN-mediated modulation of PI3K pathway was independent of the focal adhesion kinase and fatty acid synthase. The study, for the first time, established that PTEN plays a key role in the progression of pulmonary hypertension. The findings may have potential for the treatment of pulmonary hypertension using PTEN as a target.     

Single Intraperitoneal Injection of Monocrotaline as a Novel Large Animal Model of Chronic Pulmonary Hypertension in Tibet Minipigs

Objective

The purpose of this study was to establish an animal model of chronic pulmonary hypertension with a single-dose intraperitoneal injection of monocrotaline (MCT) in young Tibet minipigs, so as to enable both invasive and noninvasive measurements and hence facilitate future studies.

Methods

Twenty-four minipigs (8-week-old) were randomized to receive single-dose injection of 12.0 mg/kg MCT (MCT group, n = 12) or placebo (control group, n = 12 each). On day 42, all animals were evaluated for pulmonary hypertension with conventional transthoracic echocardiography, right heart catheterization (RHC), and pathological changes. Findings of these studies were compared between the two groups.

Results

At echocardiography, the MCT group showed significantly higher pulmonary arterial mean pressure (PAMP) compared with the controls (P<0.001). The pulmonary valve curve showed v-shaped signals with reduction of a-waves in minipigs treated with MCT. In addition, the MCT group had longer pulmonary artery pre-ejection phases, and shorter acceleration time and ejection time. RHC revealed higher mean pulmonary arterial pressure (mPAP) in the MCT group than in the control group (P<0.01). A significant and positive correlation between the mPAP values and the PAMP values (R = 0.974, P<0.0001), and a negative correlation between the mPAP and ejection time (R = 0.680, P<0.0001) was noted. Pathology demonstrated evidence of pulmonary vascular remodeling and higer index of right ventricular hypertrophy in MCT-treated minipigs.

Conclusion

A chronic pulmonary hypertension model can be successfully established in young minipigs at six weeks after MCT injection. These minipig models exhibited features of pulmonary arterial hypertension that can be evaluated by both invasive (RHC) and noninvasive (echocardiography) measurements, and may be used as an easy and stable tool for future studies on pulmonary hypertension.     

Effects of Different Pulmonary Vasodilators on Arterial Saturation in a Model of Pulmonary Hypertension

Background

Approved therapies for pulmonary arterial hypertension can induce oxygen desaturation when administered to patients with secondary forms of pulmonary hypertension (PH), probably due to an increase in ventilation/perfusion mismatch. Thus, so far these treatments have largely failed in secondary forms of PH.

Methods

We established an animal model of heterogeneous lung ventilation to evaluate the desaturation potential of mechanistically distinct vasoactive drugs launched or currently in clinical development for the treatment of PH. Single-lung ventilation was induced in five groups (N = 6) of anesthetized minipigs (7 weeks, 4 to 5 kg BW), and their hemodynamic parameters were monitored before and after intravenous injection of control (vehicle only), endothelin antagonist (bosentan; 0.3, 1, 3, 10 mg/kg), phosphodiesterase type 5 inhibitor (sildenafil; 3, 10, 30, 100 µg/kg), and soluble guanylate cyclase stimulators (BAY 41–8543 and riociguat; 1, 3, 10, 30 µg/kg). Cumulative doses were administered before successive unilateral ventilation cycles. The doses were chosen to achieve equal effect on blood pressure by the different pharmacologic principles.

Results

Single-lung ventilation resulted in transient increases in mean pulmonary artery pressure (mPAP) and desaturation. In contrast to control, all drugs dose-dependently decreased hypoxic mPAP (a positive treatment effect) and increased area under the arterial hemoglobin saturation curve (unwanted desaturation effect). Riociguat and bosentan reduced hypoxic mPAP to the greatest extent, while the soluble guanylate cyclase stimulators riociguat and BAY 41–8543 lowered arterial oxygen saturation of hemoglobin the least.

Conclusions

Future investigations will be required to confirm these findings in clinical settings.     

急性肺水肿动物模型复制

目的—探讨氯化氨诱发大鼠肺水肿动物模型效果和机理。方法—将实验大鼠随机分为对照组、氯化氨组,腹腔注射方法注入6%氯化氨(♀:6~8 ml/kg,♂:9~12 ml/kg),观察动物的一般情况、肺系数计算及病理切片,确定建立大鼠肺水肿动物模型。结果—氯化铵能引起大鼠肺水肿,其临床表现有明显特点。结论—氯化铵诱发的肺水肿模型,临床症状典型,方法简单,经济易行且重复性好,能为教学、临床、科研提供较为理想的动物模型。     

MicroRNA与动物发育

MicroRNA(miRNA)是一类约22nt大小的内源性非编码RNA,它们通过剪切靶基因的转录产物或者抑制转录产物的翻译从而起到转录后调控靶基因表达的作用。在动物体内,通过基因敲除等方法所进行的大量研究表明了miRNA参与了胚胎早期发育、脑及神经发育、心脏发育、肌肉及骨骼发育等动物发育的各个方面。miRNA是动物发生发育过程中重要的调控因子。主要介绍了近年来miRNA在动物生长发育过程中的研究进展。     

油酸诱导小型猪肺水肿模型的建立

目的制备油酸诱导小型猪肺水肿的动物模型,便于进行肺水肿的发病机制和相关治疗的研究。方法家养小型猪20只麻醉后随机分A、B两组,A组（n=10）为对照组,B组（n=10）为油酸组（油酸0.15 mL/kg经动物耳缘静脉缓慢注射）,观察两组动物肺组织病理改变、计算肺含水量及肺湿干重比。结果B组动物注射油酸后肺部出现明显的肺水肿病理改变,肺湿/干重比及肺含水量的值明显高于A组（P〈0.05）。结论本实验成功复制油酸诱导小型猪肺水肿动物模型,其病理组织切片符合肺水肿的典型病变。     

动物细胞核内miRNA的加工过程

microRNA(miRNA)是存在于真核生物中的一类大的基因家族,与其靶mRNA分子一起形成了生物体内复杂的调控网络。miRNA在基因表达调节过程中的关键性作用涉及到发育时序的控制、造血细胞的分化、细胞凋亡、细胞增殖以及器官的形成等方面。其中最值得探讨的问题是miRNA的生物发生过程及其调控机制。近年来,miRNA在动物细胞核中加工机制的研究取得了较大的进展。在细胞核中,RNA多聚酶II指导的miRNA基因的转录,微处理器作用下的pri-miRNA的剪切及exportin-5协助下的pre-miRNA的输出过程彼此协调,共同而有序的完成miRNA在细胞核中的加工过程。     

Diaphragm Atrophy and Contractile Dysfunction in a Murine Model of Pulmonary Hypertension

Pulmonary hypertension (PH) causes loss of body weight and inspiratory (diaphragm) muscle dysfunction. A model of PH induced by drug (monocrotaline, MCT) has been extensively used in mice to examine the etiology of PH. However, it is unclear if PH induced by MCT in mice reproduces the loss of body weight and diaphragm muscle dysfunction seen in patients. This is a pre-requisite for widespread use of mice to examine mechanisms of cachexia and diaphragm abnormalities in PH. Thus, we measured body and soleus muscle weight, food intake, and diaphragm contractile properties in mice after 6–8 weeks of saline (control) or MCT (600 mg/kg) injections. Body weight progressively decreased in PH mice, while food intake was similar in both groups. PH decreased (P<0.05) diaphragm maximal isometric specific force, maximal shortening velocity, and peak power. Protein carbonyls in whole-diaphragm lysates and the abundance of select myofibrillar proteins were unchanged by PH. Our findings show diaphragm isometric and isotonic contractile abnormalities in a murine model of PH induced by MCT. Overall, the murine model of PH elicited by MCT mimics loss of body weight and diaphragm muscle weakness reported in PH patients.     

Genome-wide Mechanosensitive MicroRNA (MechanomiR) Screen Uncovers Dysregulation of Their Regulatory Networks in the mdm Mouse Model of Muscular Dystrophy

Muscular dystrophies (MDs) are a heterogeneous group of genetic and neuromuscular disorders, which result in severe loss of motor ability and skeletal muscle mass and function. Aberrant mechanotransduction and dysregulated-microRNA pathways are often associated with the progression of MD. Here, we hypothesized that dysregulation of mechanosensitive microRNAs (mechanomiRs) in dystrophic skeletal muscle plays a major role in the progression of MD. To test our hypothesis, we performed a genome-wide expression profile of anisotropically regulated mechanomiRs and bioinformatically analyzed their target gene networks. We assessed their functional roles in the advancement of MD using diaphragm muscles from mdm (MD with myositis) mice, an animal model of human tibial MD (titinopathy), and their wild-type littermates. We were able to show that ex vivo anisotropic mechanical stretch significantly alters the miRNA expression profile in diaphragm muscles from WT and mdm mice; as a result, some of the genes associated with MDs are dysregulated in mdm mice due to differential regulation of a distinct set of mechanomiRs. Interestingly, we found a contrasting expression pattern of the highly expressed let-7 family mechanomiRs, let-7e-5p and miR-98–5p, and their target genes associated with the extracellular matrix and TGF-β pathways, respectively, between WT and mdm mice. Gain- and loss-of-function analysis of let-7e-5p in myocytes isolated from the diaphragms of WT and mdm mice confirmed Col1a1, Col1a2, Col3a1, Col24a1, Col27a1, Itga1, Itga4, Scd1, and Thbs1 as target genes of let-7e-5p. Furthermore, we found that miR-98 negatively regulates myoblast differentiation. Our study therefore introduces additional biological players in the regulation of skeletal muscle structure and myogenesis that may contribute to unexplained disorders of MD.     

Definition and Application of a Histopathological Scoring Scheme for an Animal Model of Acute Mycoplasma pneumoniae Pulmonary Infection

A histopathological scoring system was developed to assess the pathology of acute Mycoplasma pneumoniae pulmonary infection in a hamster model. A final score per animal (ranging 0–26) is obtained by averaging scores from each lung which have been accumulated by the addition of subscores from the assessments of quantity and quality of peribronchiolar and peribronchial infiltrates, luminal exudates, perivascular infiltrates, and parenchymal pneumonia. The scoring scheme was then applied to test the ability of a heat-killed inoculum to induce pulmonary pathology and to the trial of a 43 kDa protein-associated antigen as a vaccine immunogen. A heat-killed inoculum delivered by both intratracheal and intranasal routes did not induce pulmonary pathology compared to a live inoculum (respective mean scores 0.1, 6.7; P<0.01). Animals prevaccinated with the 43 kDa antigen developed an accentuated pathological response after live challenge compared to those unvaccinated (respective mean scores 16.8, 5.8; P=0.00007). Hypersensitization to growth medium components may, however, have contributed to the accentuated disease since the lungs of vaccinated animals challenged with culture-negative media also were affected (mean score 5.4). Reproducibility of the scoring system was measured by duplicate reading of histology slides which were randomized to the observer upon the second reading (r=0.93; P=0.000009). The scoring system has the ability to differentiate disease severity in small groups of animals.     

Monoamine Receptors in an Animal Model of Affective Disorder

After a relatively mild course of uncontrollable shocks, two distinct groups of rats can be defined in terms of their performance in learning to escape from a controllable stressor. Response-deficient (RD) rats do not learn to terminate the controllable stressor, whereas nondeficient (ND) rats learn this response as readily as do untreated control rats. The current studies were designed to determine the neurochemical correlates of the behavioral differences between these groups of rats. The major findings concerned postsynaptic beta-adrenergic effects in the hippocampus of RD rats. These included an up-regulation of beta-adrenergic receptors and, in parallel experiments, an increase in the sensitivity of adenylyl cyclase to stimulation by norepinephrine. There was no difference in brain levels of catecholamines between the three groups of rats. A statistically significant increase in levels of 5-hydroxytryptamine was noted in the hippocampus and hypothalamus of RD rats as compared to levels in ND rats, but no significant differences were measured between groups of rats in terms of S1 or S2 serotonergic receptor binding. These results implicate both beta-adrenergic and serotonergic mechanisms in the behavioral deficit caused by uncontrollable shock.     

Increased Eicosanoid Levels in the Sugen/Chronic Hypoxia Model of Severe Pulmonary Hypertension

Inflammation and altered immunity are recognized components of severe pulmonary arterial hypertension in human patients and in animal models of PAH. While eicosanoid metabolites of cyclooxygenase and lipoxygenase pathways have been identified in the lungs from pulmonary hypertensive animals their role in the pathogenesis of severe angioobliterative PAH has not been examined. Here we investigated whether a cyclooxygenase-2 (COX-2) inhibitor or diethylcarbamazine (DEC), that is known for its 5-lipoxygenase inhibiting and antioxidant actions, modify the development of PAH in the Sugen 5416/hypoxia (SuHx) rat model. The COX-2 inhibitor SC-58125 had little effect on the right ventricular pressure and did not prevent the development of pulmonary angioobliteration. In contrast, DEC blunted the muscularization of pulmonary arterioles and reduced the number of fully obliterated lung vessels. DEC treatment of SuHx rats, after the lung vascular disease had been established, reduced the degree of PAH, the number of obliterated arterioles and the degree of perivascular inflammation. We conclude that the non-specific anti-inflammatory drug DEC affects developing PAH and is partially effective once angioobliterative PAH has been established.     

Changes in Large Pulmonary Arterial Viscoelasticity in Chronic Pulmonary Hypertension

Conduit pulmonary artery (PA) stiffening is characteristic of pulmonary arterial hypertension (PAH) and is an excellent predictor of mortality due to right ventricular (RV) overload. To better understand the impact of conduit PA stiffening on RV afterload, it is critical to examine the arterial viscoelastic properties, which require measurements of elasticity (energy storage behavior) and viscosity (energy dissipation behavior). Here we hypothesize that PAH leads to frequency-dependent changes in arterial stiffness (related to elasticity) and damping ratio (related to viscosity) in large PAs. To test our hypothesis, PAH was induced by the combination of chronic hypoxia and an antiangiogenic compound (SU5416) treatment in mice. Static and sinusoidal pressure-inflation tests were performed on isolated conduit PAs at various frequencies (0.01–20 Hz) to obtain the mechanical properties in the absence of smooth muscle contraction. Static mechanical tests showed significant stiffening of large PAs with PAH, as expected. In dynamic mechanical tests, structural stiffness (κ) increased and damping ratio (D) decreased at a physiologically relevant frequency (10 Hz) in hypertensive PAs. The dynamic elastic modulus (E), a material stiffness, did not increase significantly with PAH. All dynamic mechanical properties were strong functions of frequency. In particular, κ, E and D increased with increasing frequency in control PAs. While this behavior remained for D in hypertensive PAs, it reversed for κ and E. Since these novel dynamic mechanical property changes were found in the absence of changes in smooth muscle cell content or contraction, changes in collagen and proteoglycans and their interactions are likely critical to arterial viscoelasticity in a way that has not been previously described. The impact of these changes in PA viscoelasticity on RV afterload in PAH awaits further investigation.     

内膜下血管成形术动物模型的构建

目的建立一种稳定可靠的内膜下血管成形术(subintimal angioplasty,SIA)的动物模型,进而探索术后血管壁有关组成成分的变化规律。方法以巴马猪为实验对象,一组(7只)先建立动脉硬化狭窄模型,另一组(9只)为正常动脉,在颈总动脉施行SIA,在术后不同时间观察造模段动脉的通畅率及病理变化等。结果手术操作成功率为93.8%(15/16)。SIA术后总的通畅率为53.3%(8/15)。病理组织学观察发现SIA术后动脉管壁有新内膜形成,中膜增厚,平滑肌细胞数量增多,较多泡沫细胞出现,胶原纤维及弹力纤维染色范围增大、染色密度增加。超微结构观察提示SIA术后中膜层平滑肌细胞的收缩型与合成型同时存在;细胞外基质中胶原纤维和弹力纤维含量异常丰富。结论①首次成功设计、构建内膜下血管成形术的动物模型。②内膜下血管成形术模型构建术后的病理组织学改变与腔内血管成形术后的变化基本一致。     

MicroRNA Signature Characterizes Primary Tumors That Metastasize in an Esophageal Adenocarcinoma Rat Model

Objective

To establish a miRNA signature for metastasis in an animal model of esophageal adenocarcinoma (EAC).

Background

The incidence of esophageal adenocarcinoma (EAC) has dramatically increased and esophageal cancer is now the sixth leading cause of cancer deaths worldwide. Mortality rates remain high among patients with advanced stage disease and esophagectomy is associated with high complication rates. Hence, early identification of potentially metastatic disease would better guide treatment strategies.

Methods

The modified Levrat’s surgery was performed to induce EAC in Sprague-Dawley rats. Primary EAC and distant metastatic sites were confirmed via histology and immunofluorescence. miRNA profiling was performed on primary tumors with or without metastasis. A unique subset of miRNAs expressed in primary tumors and metastases was identified with Ingenuity Pathway Analysis (IPA) along with upstream and downstream targets. miRNA-linked gene expression analysis was performed on a secondary cohort of metastasis positive (n=5) and metastasis negative (n=28) primary tumors.

Results

The epithelial origin of distant metastasis was established by IF using villin (VIL1) and mucin 5AC (MUC5AC) antibodies. miRNome analysis identified four down-regulated miRNAs in metastasis positive primary tumors compared to metastasis negative tumors: miR-92a-3p (p=0.0001), miR-141-3p (p=0.0022), miR-451-1a (p=0.0181) and miR133a-3p (p=0.0304). Six target genes identified in the top scoring networks by IPA were validated as significantly, differentially expressed in metastasis positive primary tumors: Ago2, Akt1, Kras, Bcl2L11, CDKN1B and Zeb2.

Conclusion

In vivo metastasis was confirmed in the modified Levrat’s model. Analysis of the primary tumor identified a distinctive miRNA signature for primary tumors that metastasized.