Investigation into the role of H2-Ab1 in vascular remodeling in pulmonary arterial hypertension via Bioinformatics.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a progressive disease of vascular remodeling characterized by persistent pulmonary arterial pressure elevation, which can lead to right heart failure and premature death. Given the complex pathogenesis and poor prognosis of PAH, the identification and investigation of biomarkers become increasingly critical for advancing further understanding of the disease. METHODS: PAH-related datasets, GSE49114, GSE180169 and GSE154959, were downloaded from the publicly available GEO database. By performing WGCNA on the GSE49114 dataset, a total of 906 PAH-related key module genes were screened out. By carrying out differential analysis on the GSE180169 dataset, a total of 576 differentially expressed genes were identified. Additionally, the GSE154959 single-cell sequencing dataset was also subjected to differential analysis, leading to the identification of 34 DEGs within endothelial cells. By taking intersection of the above three groups of DEGs, five PAH-related hub genes were screened out, namely Plvap, Cyp4b1, Foxf1, H2-Ab1, and H2-Eb1, among which H2-Ab1 was selected for subsequent experiments. RESULTS: A SuHx mouse model was prepared using the SU5416/hypoxia method, and the successful construction of the model was evaluated through Hematoxylin-Eosin staining, hemodynamic detection, fulton index, and Western Blot (WB). The results of WB and qRT-PCR demonstrated a significant upregulation of H2-Ab1 expression in SuHx mice. Consistent with the results of bioinformatics analysis, a time-dependent increase was observed in H2-Ab1 expression in hypoxia-treated mouse pulmonary artery endothelial cells (PAECs). To investigate whether H2-Ab1 affects the development and progression of PAH, we knocked down H2-Ab1 expression in PAECs, and found that its knockdown inhibited the viability, adhesion, migration, and angiogenesis, while concurrently promoted the apoptosis of PAECs. CONCLUSION: H2-Ab1 could regulate the proliferation, apoptosis, adhesion, migration, and angiogenesis of PAECs.

Outcomes of pregnancy in mice with pulmonary hypertension induced by Hypoxia/SU5416.

BACKGROUND: Pulmonary hypertension (PH) seriously affects the health of patients. We have found in clinical studies that PH has adverse effects on both maternal and offspring. OBJECTIVE: To establish a animal model of PH induced by hypoxia/SU5416 and observe the effects of PH on pregnant mice and their fetuses. METHODS: Twenty-four C57 mice aged 7-9 weeks were selected and divided into 4 groups with 6 mice in each group. ① Female mice with normal oxygen; ② Female mice with hypoxia/SU5416; ③ Pregnant mice with normal oxygen; ④ Pregnant mice with hypoxia/SU5416. After 19 days, weight, right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (RVHI) were compared in each group. Lung tissue and right ventricular blood were collected. The number and weight of fetal mice were also compared between the two pregnant groups. RESULTS: There was no significant difference in RVSP and RVHI between female and pregnant mice under the same condition. Compared with normal oxygen condition, two groups of mice in hypoxia/SU5416 had poor development, RVSP and RVHI were significantly increased, the number of fetal mice was small, hypoplasia, degeneration and even abortion. CONCLUSION: The model of mice PH was successfully established. PH affects the development and health of female and pregnant mice, and seriously affects the fetuses.

Inhibition of VEGFR-2 by SU5416 increases neonatally glutamate-induced neuronal damage in the cerebral motor cortex and hippocampus.

Vascular endothelial growth factor (VEGF) exerts neuroprotective or proinflammatory effects, depending on what VEGF forms (A-E), receptor types (VEGFR1-3), and intracellular signaling pathways are involved. Neonatal monosodium glutamate (MSG) treatment triggers neuronal death by excitotoxicity, which is commonly involved in different neurological disorders, including neurodegenerative diseases. This study was designed to evaluate the effects of VEGFR-2 inhibition on neuronal damage triggered by excitotoxicity in the cerebral motor cortex (CMC) and hippocampus (Hp) after neonatal MSG treatment. MSG was administered at a dose of 4 g/kg of body weight (b.w.) subcutaneously on postnatal days (PD) 1, 3, 5, and 7, whereas the VEGFR-2 inhibitor SU5416 was administered at a dose of 10 mg/kg b.w. subcutaneously on PD 5 and 7, 30 min before the MSG treatment. Neuronal damage was assessed using hematoxylin and eosin staining, fluoro-Jade staining, and TUNEL assay. Additionally, western blot assays for some proteins of the VEGF-A/VEGFR-2 signaling pathway (VEGF-A, VEGFR-2, PI3K, Akt, and iNOS) were carried out. All assays were performed on PD 6, 8, 10, and 14. Inhibition of VEGFR-2 signaling by SU5416 increases the neuronal damage induced by neonatal MSG treatment in both the CMC and Hp. Moreover, neonatal MSG treatment increased the expression levels of the studied VEGF-A/VEGFR-2 signaling pathway proteins, particularly in the CMC. We conclude that VEGF-A/VEGFR-2 signaling pathway activation could be part of the neuroprotective mechanisms that attempt to compensate for neuronal damage induced by neonatal MSG treatment and possibly also in other conditions involving excitotoxicity.

Sulforaphane prevents right ventricular injury and reduces pulmonary vascular remodeling in pulmonary arterial hypertension.

Right ventricular (RV) dysfunction is the main determinant of mortality in patients with pulmonary arterial hypertension (PAH) and while inflammation is pathogenic in PAH, there is limited information on the role of RV inflammation in PAH. Sulforaphane (SFN), a potent Nrf2 activator, has significant anti-inflammatory effects and facilitates cardiac protection in preclinical diabetic models. Therefore, we hypothesized that SFN might play a comparable role in reducing RV and pulmonary inflammation and injury in a murine PAH model. We induced PAH using SU5416 and 10% hypoxia (SuHx) for 4 wk in male mice randomized to SFN at a daily dose of 0.5 mg/kg 5 days per week for 4 wk or to vehicle control. Transthoracic echocardiography was performed to characterize chamber-specific ventricular function during PAH induction. At 4 wk, we measured RV pressure and relevant measures of histology and protein and gene expression. SuHx induced progressive RV, but not LV, diastolic and systolic dysfunction, and RV and pulmonary remodeling, fibrosis, and inflammation. SFN prevented SuHx-induced RV dysfunction and remodeling, reduced RV inflammation and fibrosis, upregulated Nrf2 expression and its downstream gene NQO1, and reduced the inflammatory mediator leucine-rich repeat and pyrin domain-containing 3 (NLRP3). SFN also reduced SuHx-induced pulmonary vascular remodeling, inflammation, and fibrosis. SFN alone had no effect on the heart or lungs. Thus, SuHx-induced RV and pulmonary dysfunction, inflammation, and fibrosis can be attenuated or prevented by SFN, supporting the rationale for further studies to investigate SFN and the role of Nrf2 and NLRP3 pathways in preclinical and clinical PAH studies.NEW & NOTEWORTHY Pulmonary arterial hypertension (PAH) in this murine model (SU5416 + hypoxia) is associated with early changes in right ventricular (RV) diastolic and systolic function. RV and lung injury in the SU5416 + hypoxia model are associated with markers for fibrosis, inflammation, and oxidative stress. Sulforaphane (SFN) alone for 4 wk has no effect on the murine heart or lungs. Sulforaphane (SFN) attenuates or prevents the RV and lung injury in the SUF5416 + hypoxia model of PAH, suggesting that Nrf2 may be a candidate target for strategies to prevent or reverse PAH.

Antiangiogenesis effect of timosaponin AIII on HUVECs in vitro and zebrafish embryos in vivo.

Timosaponin AIII (Timo AIII) is a natural steroidal saponin isolated from the traditional Chinese herb Anemarrhena asphodeloides Bge with proved effectiveness in the treatment of numerous cancers. However, whether Timo AIII suppresses tumor angiogenesis remains unclear. In the present study, we investigated the antiangiogenesis effects of Timo AIII and the underlying mechanisms in human umbilical vein endothelial cells (HUVECs) in vitro and zebrafish embryos in vivo. We showed that treatment with Timo AIII (0.5-2 µM) partially disrupted the intersegmental vessels (ISVs) and subintestinal vessels (SIVs) growth in transgenic zebrafish Tg(fli-1a: EGFP)y1. Timo AIII (0.5-4 µM) dose-dependently inhibited VEGF-induced proliferation, migration, invasion, and tube formation of HUVECs, but these inhibitory effects were not due to its cytotoxicity. We further demonstrated that Timo AIII treatment significantly suppressed the expression of VEGF receptor (VEGFR) and the phosphorylation of Akt, MEK1/2, and ERK1/2 in HUVECs. Timo AIII treatment also significantly inhibited VEGF-triggered phosphorylation of VEGFR2, Akt, and ERK1/2 in HUVECs. Moreover, we conducted RNA-Seq and analyzed the transcriptome changes in both HUVECs and zebrafish embryos following Timo AIII treatment. The coexpression network analysis results showed that various biological processes and signaling pathways were enriched including angiogenesis, cell motility, cell adhesion, protein serine/threonine kinase activity, transmembrane signaling receptor activity, growth factor activity, etc., which was consistent with the antiangiogenesis effects of Timo AIII in HUVECs and zebrafish embryos. We conclude that the antiangiogenesis effect of Timo AIII is mediated through VEGF/PI3K/Akt/MAPK signaling cascade; Timo AIII potentially exerts antiangiogenesis effect in cancer treatment.

Synthesis of Novel Class of N-Alkyl-isatin-3-iminobenzoic Acid Derivatives and Their Biological Activity in Zebrafish Embryos and Human Cancer Cell Lines.

Isatin (1H-indole-2,3-dione) and many of its derivatives are reported to have pharmacological properties. In this study, we report the synthesis and biological activity of a new class of N-alkyl-isatin-3-iminobenzoic acid derivatives prepared via the condensation of N-alkyl isatin with 4-aminobenzoic acid by conventional, microwave, and ultrasonic methods. Microwave irradiation yielded the products in a shorter reaction time with higher yields and purities. The compounds were screened in zebrafish embryos, and also in three human cancer cell lines (MCF7, HepG2, and Jurkat) and one normal human cell line i.e., human foreskin cell line (HFF-1). Two compounds (3c, 3f) were found to be highly effective against hematopoiesis in live zebrafish embryo at 10 µM concentration. The developmental stage-dependent treatment indicated that these compounds interfered with the differentiation of hemangioblasts to hematopoietic cells in zebrafish embryos. The comparative screening of semaxanib (SU5416) (a known isatin derivatives), to compounds synthesized in this study, revealed the contrasting effects of these two classes of isatin derivatives on zebrafish hematopoiesis. Most of the N-alkyl-isatin-3-iminobenzoic acid derivatives were toxic on cancer and non-cancer tested human cells lines, however, the compounds 3c and 3f specifically affected the cell viability of Jurkat cells (human hematological cell line) with least IC50 values of 16.5 and 7.8 µM. The structure-activity relationship (SAR) analysis indicated that the substitution pattern of the isatin at the 5-position was vital for activity. The in vivo and in vitro biological activities of these compounds suggested their potential use as pharmaceutical compounds for human leukemia treatment.

Endothelial cell-related autophagic pathways in Sugen/hypoxia-exposed pulmonary arterial hypertensive rats.

Pulmonary arterial hypertension (PAH) is characterized by progressive obstructive remodeling of pulmonary arteries. However, no reports have described the causative role of the autophagic pathway in pulmonary vascular endothelial cell (EC) alterations associated with PAH. This study investigated the time-dependent role of the autophagic pathway in pulmonary vascular ECs and pulmonary vascular EC kinesis in a severe PAH rat model (Sugen/hypoxia rat) and evaluated whether timely induction of the autophagic pathway by rapamycin improves PAH. Hemodynamic and histological examinations as well as flow cytometry of pulmonary vascular EC-related autophagic pathways and pulmonary vascular EC kinetics in lung cell suspensions were performed. The time-dependent and therapeutic effects of rapamycin on the autophagic pathway were also assessed. Sugen/hypoxia rats treated with the vascular endothelial growth factor receptor blocker SU5416 showed increased right ventricular systolic pressure (RVSP) and numbers of obstructive vessels due to increased pulmonary vascular remodeling. The expression of the autophagic marker LC3 in ECs also changed in a time-dependent manner, in parallel with proliferation and apoptotic markers as assessed by flow cytometry. These results suggest the presence of cross talk between pulmonary vascular remodeling and the autophagic pathway, especially in small vascular lesions. Moreover, treatment of Sugen/hypoxia rats with rapamycin after SU5416 injection activated the autophagic pathway and improved the balance between cell proliferation and apoptosis in pulmonary vascular ECs to reduce RVSP and pulmonary vascular remodeling. These results suggested that the autophagic pathway can suppress PAH progression and that rapamycin-dependent activation of the autophagic pathway could ameliorate PAH.

Animal models of pulmonary arterial hypertension: A systematic review and meta-analysis of data from 6126 animals.

Numerous animal models of pulmonary hypertension are currently available. A systematic review and meta-analysis was performed of a number of experimental studies of disease induction based on several animal models. A meta-analysis was performed of 291 publications discussing the efficacy of 611 interventions to introduce disease pulmonary hypertension in 6126 animals. A meta-regression analysis was done to assess the effect of prolonged periods of disease induction on the outcomes. A random-effects meta-analysis was used to assess the impact of study characteristics and seek evidence of publication bias. A more pronounced worsening in hemodynamics or right ventricle hypertrophy was observed in animals exposed to Sugen combined with hypoxia, or left pneumonectomy followed by monocrotaline. Chronic hypoxia induced the poorest, but the most stable, response to disease induction with regard to elevated hemodynamic parameters, right ventricle hypertrophy and wall thickening. The greatest elevation of right ventricle systolic pressure was observed in animals exposed to isoflurane and the weakest to chloral hydrate. This result was true for different animal models and lengths of induction of pulmonary hypertension. Publication bias was found for all the crucial parameters. Development of pulmonary hypertension depends on the choice of animal model. Classic models, especially these related to chronic hypoxia, provoke a less severe response with regard to poorer hemodynamics and myocardial hypertrophy. The outcome of disease development can be strongly determined by the duration of induction, detailed experimental conditions and anesthesia procedure.

Marked Strain-Specific Differences in the SU5416 Rat Model of Severe Pulmonary Arterial Hypertension.

We assessed the pulmonary hemodynamic response to vascular endothelial growth factor receptor, type 2, inhibition using SU5416 (SU) with and without chronic hypoxia (CH) in different background strains and colonies of rats. A single subcutaneous injection of SU (20 mg/kg) or vehicle was administered to different substrains of Sprague-Dawley (SD) rats, and they were compared with Lewis and Fischer rats, with and without exposure to CH (10% O2 for 3 wk). Remarkably, a unique colony of SD rats from Charles River Laboratories, termed the SD-hyperresponsive type, exhibited severe pulmonary arterial hypertension (PAH) with SU alone, characterized by increased right ventricular systolic pressure, right ventricular/left ventricular plus septal weight ratio, and arteriolar occlusive lesions at 7-8 weeks (all P < 0.0001 versus vehicle). In contrast, the other SD substrain from Harlan Laboratories, termed SD-typical type, as well as Fischer rats, developed severe PAH only when exposed to SU and CH, whereas Lewis rats showed only a minimal response. All SD-typical type rats survived for up to 13 weeks after SU/CH, whereas SD-hyperresponsive type rats exhibited mortality after SU and SU/CH (35% and 50%, respectively) at 8 weeks. Fischer rats exposed to SU/CH exhibited the greatest mortality at 8 weeks (78%), beginning as early as 4 weeks after SU and preceded by right ventricle enlargement. Of note, a partial recovery of PAH after 8 weeks was observed in the SD-typical type substrain only. In conclusion, variation in strain, even between colonies of the same strain, has a remarkable influence on the nature and severity of the response to SU, consistent with an important role for genetic modifiers of the PAH phenotype.

Pneumonectomy combined with SU5416 induces severe pulmonary hypertension in rats.

The SU5416 + hypoxia (SuHx) rat model is a commonly used model of severe pulmonary arterial hypertension. While it is known that exposure to hypoxia can be replaced by another type of hit (e.g., ovalbumin sensitization) it is unknown whether abnormal pulmonary blood flow (PBF), which has long been known to invoke pathological changes in the pulmonary vasculature, can replace the hypoxic exposure. Here we studied if a combination of SU5416 administration combined with pneumonectomy (PNx), to induce abnormal PBF in the contralateral lung, is sufficient to induce severe pulmonary arterial hypertension (PAH) in rats. Sprague Dawley rats were subjected to SuPNx protocol (SU5416 + combined with left pneumonectomy) or standard SuHx protocol, and comparisons between models were made at week 2 and 6 postinitiation. Both SuHx and SuPNx models displayed extensive obliterative vascular remodeling leading to an increased right ventricular systolic pressure at week 6 Similar inflammatory response in the lung vasculature of both models was observed alongside increased endothelial cell proliferation and apoptosis. This study describes the SuPNx model, which features severe PAH at 6 wk and could serve as an alternative to the SuHx model. Our study, together with previous studies on experimental models of pulmonary hypertension, shows that the typical histopathological findings of PAH, including obliterative lesions, inflammation, increased cell turnover, and ongoing apoptosis, represent a final common pathway of a disease that can evolve as a consequence of a variety of insults to the lung vasculature.

Synthesis and preliminary evaluation of 5,7-dimethyl-2-aryl-3H-pyrrolizin-3-ones as angiogenesis inhibitors.

Sunitinib (Sutent®) is a receptor tyrosine kinase (RTK) and angiogenesis inhibitor approved for the treatment of renal cell carcinomas, gastrointestinal stromal tumours and pancreatic neuroendocrine tumours. A key structural motif retained throughout medicinal chemistry efforts during sunitinib's development was the indoline-2-one group. In the search for new anti-angiogenic scaffolds, we previously reported that non-indoline-2-one-based derivatives of semaxanib (SU5416, a structurally simpler sunitinib predecessor that underwent Phase III trials) are active as angiogenesis inhibitors, indicating that the group is not essential for activity. This Letter describes the synthesis and structure-activity relationships of another class of non-indoline-2-one angiogenesis inhibitors related to sunitinib/semaxanib; the 5,7-dimethyl-2-aryl-3H-pyrrolizin-3-ones. A focussed library of 19 analogues was prepared using a simple novel process, wherein commercially available substituted arylacetic acids activated with an amide coupling reagent (HBTU) were reacted with the potassium salt of 3,5-dimethyl-1H-pyrrole-2-carbaldehyde in one-pot. Screening of the library using a cell-based endothelial tube formation assay identified 6 compounds with anti-angiogenesis activity. Two of the compounds were advanced to the more physiologically relevant rat aortic ring assay, where they showed similar inhibitory effects to semaxanib at 10µg/mL, confirming that 5,7-dimethyl-2-aryl-3H-pyrrolizin-3-ones represent a new class of angiogenesis inhibitors.

Targeted activation of endothelin-1 exacerbates hypoxia-induced pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a fatal disease that eventually results in right heart failure and death. Current pharmacologic therapies for PAH are limited, and there are no drugs that could completely cure PAH. Enhanced activity of endothelin system has been implicated in PAH severity and endothelin receptor antagonists have been used clinically to treat PAH. However, there is limited experimental evidence on the direct role of enhanced endothelin system activity in PAH. Here, we investigated the correlation between endothelin-1 (ET-1) and PAH using ET-1 transgenic (ETTG) mice. Exposure to chronic hypoxia increased right ventricular pressure and pulmonary arterial wall thickness in ETTG mice compared to those in wild type mice. Of note, ETTG mice exhibited modest but significant increase in right ventricular pressure and vessel wall thickness relative to wild type mice even under normoxic conditions. To induce severe PAH, we administered SU5416, a vascular endothelial growth factor receptor inhibitor, combined with exposure to chronic hypoxia. Treatment with SU5416 modestly aggravated hypoxia-induced pulmonary hypertension, right ventricular hypertrophy, and pulmonary arterial vessel wall thickening in ETTG mice in association with increased interleukin-6 expression in blood vessels. However, there was no sign of obliterative endothelial cell proliferation and plexiform lesion formation in the lungs. These results demonstrated that enhanced endothelin system activity could be a causative factor in the development of PAH and provided rationale for the inhibition of endothelin system to treat PAH.

ID3 contributes to the acquisition of molecular stem cell-like signature in microvascular endothelial cells: its implication for understanding microvascular diseases.

While significant progress has been made to advance our knowledge of microvascular lesion formation, yet the investigation of how stem-like cells may contribute to the pathogenesis of microvascular diseases is still in its infancy. We assessed whether the inhibitor of DNA binding and differentiation 3 (ID3) contributes to the acquisition of a molecular stem cell-like signature in microvascular endothelial cells. The effects of stable ID3 overexpression and SU5416 treatment - a chemical inducer of microvascular lesions, had on the stemness signature were determined by flow cytometry, immunoblot, and immunohistochemistry. Continuous ID3 expression produced a molecular stemness signature consisting of CD133(+) VEGFR3(+) CD34(+) cells. Cells exposed to SU5416 showed positive protein expression of ID3, VEGFR3, CD34 and increased expression of pluripotent transcription factors Oct-4 and Sox-2. ID3 overexpressing cells supported the formation of a 3-D microvascular lesion co-cultured with smooth muscle cells. In addition, in vivo microvascular lesions from SuHx rodent model showed an increased expression of ID3, VEGFR3, and Pyk2 similar to SU5416 treated human endothelial cells. Further investigations into how normal and stem-like cells utilize ID3 may open up new avenues for a better understanding of the molecular mechanisms which are underlying the pathological development of microvascular diseases.

Hypoxic stress, brain vascular system, and ß-amyloid: a primary cell culture study.

This study stresses the hypothesis whether hypoxic events contribute to formation and deposition of ß-amyloid (Aß) in cerebral blood vessels by affecting the processing of endothelial amyloid precursor protein (APP). Therefore, cerebral endothelial cells (ECs) derived from transgenic Tg2576 mouse brain, were subjected to short periods of hypoxic stress, followed by assessment of formation and secretion of APP cleavage products sAPPα, sAPPß, and Aß as well as the expression of endothelial APP. Hypoxic stress of EC leads to enhanced secretion of sAPPß into the culture medium as compared to normoxic controls, which is accompanied by increased APP expression, induction of vascular endothelial growth factor (VEGF) synthesis, nitric oxide production, and differential changes in endothelial p42/44 (ERK1/2) expression. The hypoxia-mediated up-regulation of p42/44 at a particular time of incubation was accompanied by a corresponding down-regulation of the phosphorylated form of p42/44. To reveal any role of hypoxia-induced VEGF in endothelial APP processing, ECs were exposed by VEGF. VEGF hardly affected the amount of sAPPß and Aß(1-40) secreted into the culture medium, whereas the suppression of the VEGF receptor action by SU-5416 resulted in decreased release of sAPPß and Aß(1-40) in comparison to control incubations, suggesting a role of VEGF in controlling the activity of γ-secretase, presumably via the VEGF receptor-associated tyrosine kinase. The data suggest that hypoxic stress represents a mayor risk factor in causing Aß deposition in the brain vascular system by favoring the amyloidogenic route of endothelial APP processing. The hypoxic-stress-induced changes in ß-secretase activity are presumably mediated by altering the phosphorylation status of p42/44, whereas the stress-induced up-regulation of VEGF appears to play a counteracting role by maintaining the balance of physiological APP processing.

The role of vascular endothelial growth factor in pulmonary arterial hypertension. The angiogenesis paradox.

Pulmonary arterial hypertension (PAH) is characterized by dysfunctional angiogenesis leading to lung vessel obliteration. PAH is widely considered a proangiogenic disease; however, the role of angiogenic factors, such as the vascular endothelial growth factor (VEGF) and its receptors, in the pathobiology of PAH remains incompletely understood. This Review attempts to untangle some of the complex multilayered actions of VEGF to provide a VEGF-centered perspective of PAH. Furthermore, we provide a cogent explanation for the paradox of VEGF receptor blockade-induced pulmonary hypertension that characterizes the SU5416-hypoxia rat model of PAH, and attempt to translate the knowledge gained from the experimental model to the human disease by postulating the potential role of endogenous (SU5416-like) VEGF inhibitors. The main objective of this Review is to promote discussion and investigation of the opposing and complementary actions of VEGF in PAH. Understanding the balance between angiogenic and antiangiogenic factors and their role in the pathogenesis of PAH will be necessary before antiangiogenic drugs can be considered for the treatment of PAH.

Unilateral Lung Removal in Combination with Monocrotaline or SU5416 in Rodents: A Reliable Model to Mimic the Pathology of the Human Pulmonary Hypertension.

Pulmonary hypertension (PH) is a chronic and progressive disorder characterized by elevated mean pulmonary arterial pressure, pulmonary vascular remodeling, and the development of concentric laminar intimal fibrosis with plexiform lesions. While rodent models have been developed to study PH, they have certain deficiencies and do not entirely replicate the human disease due to the heterogeneity of PH pathology. Therefore, combined models are necessary to study PH. Recent studies have shown that altered pulmonary blood flow is a significant trigger in the development of vascular remodeling and neointimal lesions. One of the most promising rodent models for increased pulmonary flow is the combination of unilateral left pneumonectomy with a "second hit" of monocrotaline (MCT) or SU5416. The removal of one lung in this model forces blood to circulate only in the other lung and induces increased and turbulent pulmonary blood flow. This increased vascular flow leads to progressive remodeling and occlusion of small pulmonary arteries. The second hit by MCT or SU5416 leads to endothelial cell dysfunction, resulting in severe PH and the development of plexiform arteriopathy.

Neuropilin-1 modulates vascular endothelial growth factor-induced poly(ADP-ribose)-polymerase leading to reduced cerebrovascular apoptosis.

Cerebral ischemia is encompassed by cerebrovascular apoptosis, yet the mechanisms behind apoptosis regulation are not fully understood. We previously demonstrated inhibition of endothelial apoptosis by vascular endothelial growth factor (VEGF) through upregulation of poly(ADP-ribose)-polymerase (PARP) expression. However, PARP overactivation through oxidative stress can lead to necrosis. This study tested the hypothesis that neuropilin-1 (NP-1), an alternative VEGF receptor, regulates the response to cerebral ischemia by modulating PARP expression and, in turn, apoptosis inhibition by VEGF. In endothelial cell culture, NP-1 colocalized with VEGF receptor-2 (VEGFR-2) and acted as its coreceptor. This significantly enhanced VEGF-induced PARP mRNA and protein expression demonstrated by receptor-specific inhibitors and VEGF-A isoforms. NP-1 augmented the inhibitory effect of VEGF/VEGFR-2 interaction on apoptosis induced by adhesion inhibition through the αV-integrin inhibitor cRGDfV. NP-1/VEGFR-2 signal transduction involved JNK and Akt. In rat models of permanent and temporary middle cerebral artery occlusion, the ischemic cerebral hemispheres displayed endothelial and neuronal apoptosis next to increased endothelial NP-1 and VEGFR-2 expression compared to non-ischemic cerebral hemispheres, sham-operated or untreated controls. Increased vascular superoxide dismutase-1 and catalase expression as well as decreased glycogen reserves indicated oxidative stress in the ischemic brain. Of note, protein levels of intact PARP remained stable despite pro-apoptotic conditions through increased PARP mRNA production during cerebral ischemia. In conclusion, NP-1 is upregulated in conditions of imminent cerebrovascular apoptosis to reinforce apoptosis inhibition and modulate VEGF-dependent PARP expression and activation. We propose that NP-1 is a key modulator of VEGF maintaining cerebrovascular integrity during ischemia. Modulating the function of NP-1 to target PARP could help to prevent cellular damage in cerebrovascular disease.

Dehydroepiandrosterone restores right ventricular structure and function in rats with severe pulmonary arterial hypertension.

Current therapy of pulmonary arterial hypertension (PAH) is inadequate. Dehydroepiandrosterone (DHEA) effectively treats experimental pulmonary hypertension in chronically hypoxic and monocrotaline-injected rats. Contrary to these animal models, SU5416/hypoxia/normoxia-exposed rats develop a more severe form of occlusive pulmonary arteriopathy and right ventricular (RV) dysfunction that is indistinguishable from the human disorder. Thus, we tested the effects of DHEA treatment on PAH and RV structure and function in this model. Chronic (5 wk) DHEA treatment significantly, but moderately, reduced the severely elevated RV systolic pressure. In contrast, it restored the impaired cardiac index to normal levels, resulting in an improved cardiac function, as assessed by echocardiography. Moreover, DHEA treatment inhibited RV capillary rarefaction, apoptosis, fibrosis, and oxidative stress. The steroid decreased NADPH levels in the RV. As a result, the reduced reactive oxygen species production in the RV of these rats was reversed by NADPH supplementation. Mechanistically, DHEA reduced the expression and activity of Rho kinases in the RV, which was associated with the inhibition of cardiac remodeling-related transcription factors STAT3 and NFATc3. These results show that DHEA treatment slowed the progression of severe PAH in SU5416/hypoxia/normoxia-exposed rats and protected the RV against apoptosis and fibrosis, thus preserving its contractile function. The antioxidant activity of DHEA, by depleting NADPH, plays a central role in these cardioprotective effects.

Expression and localization of vascular endothelial growth factor and its receptors in the pig uterus during peri-implantation and determination of the in vitro effect of the angiogenesis inhibitor SU5416 on VEGF system expression.

The aim of this work was to determine endometrial mRNA expression and uterine protein localization of vascular endothelial growth factor (VEGF) and its receptors VEGFR1 and VEGFR2 during the estrous cycle and peri-implantation period in sows. Uterine tissues were collected from pregnant sows on days 12, 14, 16, and 18 after artificial insemination and from non-pregnant animals on days 2 and 12 of the estrous cycle (day 0 = day of estrus). Using immunohistochemistry, a positive signal for VEGF and its receptor VEGFR2 was found in uterine luminal epithelial cells, endometrial glands, stroma, blood vessels, and myometrium. A VEGFR1 signal was only found in endometrial and myometrial blood vessels and stroma. By day 18 of gestation, the mRNA expression levels of VEGF, VEGFR1, and VEGFR2 were higher than those observed on days 2 and 12 of the estrous cycle and on days 12, 14, and 16 of gestation. Then, a primary culture of sow endometrial epithelial cells was established to define the potential of the selective inhibition of VEGFR2 after treatment with inhibitor SU5416 and determine its effects on the expression pattern of the VEGF system. The endometrial epithelial cells treated with SU5416 showed a dose-dependent decrease in VEGFR1 and VEGFR2 mRNA expression. The present study provides additional evidence on the importance of the VEGF system during peri-implantation, as well as on the specific inhibitory activity of SU5416 in epithelial cells, which, as demonstrated, express the protein and mRNA of VEGF and its receptors VEGFR1 and VEGFR2.