VIP and endothelin receptor antagonist: An effective combination against experimental pulmonary arterial hypertension

Background

Pulmonary Arterial Hypertension (PAH) remains a therapeutic challenge, and the search continues for more effective drugs and drug combinations. We recently reported that deletion of the vasoactive intestinal peptide (VIP) gene caused the spontaneous expression of a PH phenotype that was fully corrected by VIP. The objectives of this investigation were to answer the questions: 1) Can VIP protect against PH in other experimental models? and 2) Does combining VIP with an endothelin (ET) receptor antagonist bosentan enhance its efficacy?

Methods

Within 3 weeks of a single injection of monocrotaline (MCT, s.c.) in Sprague Dawley rats, PAH developed, manifested by pulmonary vascular remodeling, lung inflammation, RV hypertrophy, and death within the next 2 weeks. MCT-injected animals were either untreated, treated with bosentan (p.o.) alone, with VIP (i.p.) alone, or with both together. We selected this particular combination upon finding that VIP down-regulates endothelin receptor expression which is further suppressed by bosentan. Therapeutic outcomes were compared as to hemodynamics, pulmonary vascular pathology, and survival.

Results

Treatment with VIP, every other day for 3 weeks, begun on the same day as MCT, almost totally prevented PAH pathology, and eliminated mortality for 45 days. Begun 3 weeks after MCT, however, VIP only partially reversed PAH pathology, though more effectively than bosentan. Combined therapy with both drugs fully reversed the pathology, while preventing mortality for at least 45 days.

Conclusions

1) VIP completely prevented and significantly reversed MCT-induced PAH; 2) VIP was more effective than bosentan, probably because it targets a wider range of pro-remodeling pathways; and 3) combination therapy with VIP plus bosentan was more effective than either drug alone, probably because both drugs synergistically suppressed ET-ET receptor pathway.     

Chronic thromboembolic pulmonary hypertension

Chronic pulmonary thromboembolic disease is an important cause of severe pulmonary hypertension, and as such is associated with significant morbidity and mortality. The prognosis of this condition reflects the degree of associated right ventricular dysfunction, with predictable mortality related to the severity of the underlying pulmonary hypertension. Left untreated, the prognosis is poor. Pulmonary endarterectomy is the treatment of choice to relieve pulmonary artery obstruction in patients with chronic thromboembolic pulmonary hypertension and has been remarkably successful. Advances in surgical techniques along with the introduction of pulmonary hypertension-specific medication provide therapeutic options for the majority of patients afflicted with the disease. However, a substantial number of patients are not candidates for pulmonary endarterectomy due to either distal pulmonary vascular obstruction or significant comorbidities. Therefore, careful selection of surgical candidates in expert centres is paramount. The current review focuses on the diagnostic approach to chronic thromboembolic pulmonary hypertension and the available surgical and medical therapeutic options.

Electronic supplementary material

The online version of this article (doi:10.1007/s12471-014-0592-2) contains supplementary material, which is available to authorized users.     

Anxiety and depression disorders in patients with pulmonary arterial hypertension and chronic thromboembolic pulmonary hypertension

Background

The objective of this prospective study was to assess the prevalence of anxiety and depression disorders and their association with quality of life (QoL), clinical parameters and survival in patients with pulmonary hypertension (PH).

Methods

We prospectively assessed 158 patients invasively diagnosed with pulmonary arterial hypertension (n = 138) and inoperable chronic thromboembolic PH (n = 20) by clinical measures including quality of life (QoL, SF-36 questionnaire), cardiopulmonary exercise testing and six minute walking distance and by questionnaires for depression (PHQ-9) and anxiety (GAD-7). According to the results of the clinical examination and the questionnaires for mental disorders (MD) patients were classified into two groups, 1) with moderate to severe MD (n = 36, 22,8%), and 2) with mild or no MD (n = 122). Patients were followed for a median of 2.7 years. Investigators of QoL, SF-36 were blinded to the clinical data.

Results

At baseline the 2 groups did not differ in their severity of PH or exercise capacity. Patients with moderate to severe MD (group 1) had a significantly lower QoL shown in all subscales of SF-36 (p < 0.002). QoL impairment significantly correlated with the severity of depression (p < 0.001) and anxiety (p < 0.05). During follow-up period 32 patients died and 3 were lost to follow-up. There was no significant difference between groups regarding survival. Only 8% of the patients with MD received psychopharmacological treatment.

Conclusion

Anxiety and depression were frequently diagnosed in our patients and significantly correlated with quality of life, but not with long term survival. Further prospective studies are needed to confirm the results.     

The MicroRNA-130/301 Family Controls Vasoconstriction in Pulmonary Hypertension

Pulmonary hypertension (PH) is a complex disorder, spanning several known vascular cell types. Recently, we identified the microRNA-130/301 (miR-130/301) family as a regulator of multiple pro-proliferative pathways in PH, but the true breadth of influence of the miR-130/301 family across cell types in PH may be even more extensive. Here, we employed targeted network theory to identify additional pathogenic pathways regulated by miR-130/301, including those involving vasomotor tone. Guided by these predictions, we demonstrated, via gain- and loss-of-function experimentation in vitro and in vivo, that miR-130/301-specific control of the peroxisome proliferator-activated receptor γ regulates a panel of vasoactive factors communicating between diseased pulmonary vascular endothelial and smooth muscle cells. Of these, the vasoconstrictive factor endothelin-1 serves as an integral point of communication between the miR-130/301-peroxisome proliferator-activated receptor γ axis in endothelial cells and contractile function in smooth muscle cells. Thus, resulting from an in silico analysis of the architecture of the PH disease gene network coupled with molecular experimentation in vivo, these findings clarify the expanded role of the miR-130/301 family in the global regulation of PH. They further emphasize the importance of molecular cross-talk among the diverse cellular populations involved in PH.     

Differences of cardiac output measurements by open-circuit acetylene uptake in pulmonary arterial hypertension and chronic thromboembolic pulmonary hypertension: a cohort study

Background

As differences in gas exchange between pulmonary arterial hypertension (PAH) and chronic thromboembolic pulmonary hypertension (CTEPH) have been demonstrated, we asked if cardiac output measurements determined by acetylene (C₂H₂) uptake significantly differed in these diseases when compared to the thermodilution technique.

Method

Single-breath open-circuit C₂H₂ uptake, thermodilution, and cardiopulmonary exercise testing were performed in 72 PAH and 32 CTEPH patients.

Results

In PAH patients the results for cardiac output obtained by the two methods showed an acceptable agreement with a mean difference of -0.16 L/min (95% CI -2.64 to 2.32 L/min). In contrast, the agreement was poorer in the CTEPH group with the difference being -0.56 L/min (95% CI -4.96 to 3.84 L/min). Functional dead space ventilation (44.5 ± 1.6 vs. 32.2 ± 1.4%, p < 0.001) and the mean arterial to end-tidal CO₂ gradient (9.9 ± 0.8 vs. 4.1 ± 0.5 mmHg, p < 0.001) were significantly elevated among CTEPH patients.

Conclusion

Cardiac output evaluation by the C₂H₂ technique should be interpreted with caution in CTEPH, as ventilation to perfusion mismatching might be more relevant than in PAH.     

Clinical worsening after pulmonary endarterectomy in chronic thromboembolic pulmonary hypertension

Introduction

Pulmonary endarterectomy (PEA) is the most effective treatment for chronic thromboembolic pulmonary hypertension (CTEPH). The aim of this study is to evaluate long-term survival and freedom from clinical worsening after PEA.

Methods

All patients who underwent PEA in our hospital between May 2000 and August 2009 were included. Follow-up parameters were all-cause mortality and time to clinical worsening, defined as a combination of death, need for pulmonary hypertension-specific medication or 15% decrease in six-minute walk distance without improvement in functional class. The Cox proportional hazard regression was used to identify predictors.

Results

Seventy-four consecutive patients (mean age 55.9 ± 13.8 years, 51% female) underwent PEA. Prior to surgery, 55 patients were in NYHA functional class III or higher. The mean pulmonary artery pressure was 41.3 ± 11.9 mmHg with a mean pulmonary vascular resistance of 521 ± 264 dyn·s·cm⁻⁵ (range 279–1331 dyn·s·cm⁻⁵). Five patients (6.8%) died in-hospital. Out of hospital, 5 out of 69 patients (7.2%) died during a median follow-up of 3.7 ± 2.2 years [range 0.1–8.5 years]). The one- and five-year survival rates were 93% and 89%, respectively. During follow-up, clinical worsening occurred in 13 out of 69 patients (18.8%). The one- and five-year rates of freedom from clinical worsening were 94% and 72%, respectively. The baseline NT-pro BNP level tended to be a predictor for occurrence of clinical worsening.

Conclusion

Pulmonary endarterectomy is associated with good long-term survival in patients with CTEPH. However, clinical worsening occurred in a substantial number of patients at long-term follow-up.     

A low resting heart rate at diagnosis predicts favourable long-term outcome in pulmonary arterial and chronic thromboembolic pulmonary hypertension. A prospective observational study

Background

A low resting heart rate (HR) is prognostically favourable in healthy individuals and in patients with left heart disease. In this study we investigated the impact of HR at diagnosis on long-term outcome in patients with differently classified precapillary pulmonary hypertension (pPH).

Methods

pPH patients diagnosed as pulmonary arterial (PAH) or inoperable chronic thromboembolic pulmonary hypertension (CTEPH) were registered and regularly followed at our centre Baseline characteristics and events defined as either death or lung transplantation were noted. The prognostic value of HR was analysed using Kaplan Meier estimates, live tables and Cox regression.

Results

206 patients with PAH (148) and inoperable CTEPH (58) were included. The median HR was 82 bpm. pPH with a HR below 82 bpm had a significantly longer overall event-free survival (2409 vs.1332 days, p = .000). This advantage was similarly found if PAH and CTEPH were analysed separately. Although a lower HR was associated with a better hemodynamic and functional class, HR was a strong and independent prognostic marker for transplant free survival even if corrected for age, sex, hemodynamics and functional status.

Conclusion

We show that resting HR at diagnosis is a strong and independent long-term prognostic marker in PAH and CTEPH. Whether reducing HR by pharmacological agents would improve outcome in pPH has to be assessed by future trials with high attention to safety.     

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.     

Endothelial-like cells in chronic thromboembolic pulmonary hypertension: crosstalk with myofibroblast-like cells

Background

Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by intravascular thrombus formation in the pulmonary arteries.Recently, it has been shown that a myofibroblast cell phenotype was predominant within endarterectomized tissues from CTEPH patients. Indeed, our recent study demonstrated the existence of not only myofibroblast-like cells (MFLCs), but also endothelial-like cells (ELCs). Under in vitro conditions, a few transitional cells (co-expressing both endothelial- and SM-cell markers) were observed in the ELC population. We hypothesized that MFLCs in the microenvironment created by the unresolved clot may promote the endothelial-mesenchymal transition and/or induce endothelial cell (EC) dysfunction.

Methods

We isolated cells from these tissues and identified them as MFLCs and ELCs. In order to test whether the MFLCs provide the microenvironment which causes EC alterations, ECs were incubated in serum-free medium conditioned by MFLCs, or were grown in co-culture with the MFLCs.

Results

Our experiments demonstrated that MFLCs promoted the commercially available ECs to transit to other mesenchymal phenotypes and/or induced EC dysfunction through inactivation of autophagy, disruption of the mitochondrial reticulum, alteration of the SOD-2 localization, and decreased ROS production. Indeed, ELCs included a few transitional cells, lost the ability to form autophagosomes, and had defective mitochondrial structure/function. Moreover, rapamycin reversed the phenotypic alterations and the gene expression changes in ECs co-cultured with MFLCs, thus suggesting that this agent had beneficial therapeutic effects on ECs in CTEPH tissues.

Conclusions

It is possible that the microenvironment created by the stabilized clot stimulates MFLCs to induce EC alterations.     

Cellular mechanosignaling in pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a vasculopathy characterized by sustained elevated pulmonary arterial pressures in which the pulmonary vasculature undergoes significant structural and functional remodeling. To better understand disease mechanisms, in this review article we highlight recent insights into the regulation of pulmonary arterial cells by mechanical cues associated with PAH. Specifically, the mechanobiology of pulmonary arterial endothelial cells (PAECs), smooth muscle cells (PASMCs) and adventitial fibroblasts (PAAFs) has been investigated in vivo, in vitro, and in silico. Increased pulmonary arterial pressure increases vessel wall stress and strain and endothelial fluid shear stress. These mechanical cues promote vasoconstriction and fibrosis that contribute further to hypertension and alter the mechanical milieu and regulation of pulmonary arterial cells.     

Determinants of an elevated pulmonary arterial pressure in patients with pulmonary arterial hypertension

Given the difficulty of diagnosing early-stage pulmonary arterial hypertension (PAH) due to the lack of signs and symptoms, and the risk of an open lung biopsy, the precise pathological features of presymptomatic stage lung tissue remain unknown. It has been suggested that the maximum elevation of the mean pulmonary arterial pressure (P_pa) is achieved during the early symptomatic stage, indicating that the elevation of the mean P_pa is primarily driven by the pulmonary vascular tone and/or some degree of pulmonary vascular remodeling completed during this stage. Recently, the examination of a rat model of severe PAH suggested that the severe PAH may be primarily determined by the presence of intimal lesions and/or the vascular tone in the early stage. Human data seem to indicate that intimal lesions are essential for the severely increased pulmonary arterial blood pressure in the late stage of the disease.However, many questions remain. For instance, how does the pulmonary hemodynamics change during the course of the disease, and what drives the development of severe PAH? Although it is generally acknowledged that both pulmonary vascular remodeling and the vascular tone are important determinants of an elevated pulmonary arterial pressure, which is the root cause of the time-dependent progression of the disease? Here we review the recent histopathological concepts of PAH with respect to the progression of the lung vascular disease.     

510.6nm激光照射对兔血管平滑肌细胞增殖的影响

本文用５１０．６ｎｍ 波长激光以功率密度１、５、１０ ｍ Ｗ／ｃｍ ２ 和能量密度２、４、６Ｊ／ｃｍ ２ 照射体外培养的兔血管平滑肌细胞（ＳＭＣ）,通过３Ｈ－ ＴｄＲ掺入率和细胞生长曲线测定细胞增殖率。结果显示,上述激光照射量均能抑制细胞增殖率,其中以１０ｍ Ｗ／ｃｍ ２ 组的作用最为显著     

CD8 positive T cells express IL-17 in patients with chronic obstructive pulmonary disease

Background

Chronic obstructive pulmonary disease (COPD) is a progressive and irreversible chronic inflammatory disease of the lung. The nature of the immune reaction in COPD raises the possibility that IL-17 and related cytokines may contribute to this disorder. This study analyzed the expression of IL-17A and IL-17F as well as the phenotype of cells producing them in bronchial biopsies from COPD patients.

Methods

Bronchoscopic biopsies of the airway were obtained from 16 COPD subjects (GOLD stage 1-4) and 15 control subjects. Paraffin sections were used for the investigation of IL-17A and IL-17F expression in the airways by immunohistochemistry, and frozen sections were used for the immunofluorescence double staining of IL-17A or IL-17F paired with CD4 or CD8. In order to confirm the expression of IL-17A and IL-17F at the mRNA level, a quantitative RT-PCR was performed on the total mRNA extracted from entire section or CD8 positive cells selected by laser capture microdissection.

Results

IL-17F immunoreactivity was significantly higher in the bronchial biopsies of COPD patients compared to control subjects (P < 0.0001). In the submucosa, the absolute number of both IL-17A and IL-17F positive cells was higher in COPD patients (P < 0.0001). After adjusting for the total number of cells in the submucosa, we still found that more cells were positive for both IL-17A (P < 0.0001) and IL-17F (P < 0.0001) in COPD patients compared to controls. The mRNA expression of IL-17A and IL-17F in airways of COPD patients was confirmed by RT-PCR. The expression of IL-17A and IL-17F was co-localized with not only CD4 but also CD8, which was further confirmed by RT-PCR on laser capture microdissection selected CD8 positive cells.

Conclusion

These findings support the notion that Th17 cytokines could play important roles in the pathogenesis of COPD, raising the possibility of using this mechanism as the basis for novel therapeutic approaches.     

Epigenetics of hypoxic pulmonary arterial hypertension following intrauterine growth retardation rat: epigenetics in PAH following IUGR

Background

Accumulating evidence reveals that intrauterine growth retardation (IUGR) can cause varying degrees of pulmonary arterial hypertension (PAH) later in life. Moreover, epigenetics plays an important role in the fetal origin of adult disease. The goal of this study was to investigate the role of epigenetics in the development of PAH following IUGR.

Methods

The IUGR rats were established by maternal undernutrition during pregnancy. Pulmonary vascular endothelial cells (PVEC) were isolated from the rat lungs by magnetic-activated cell sorting (MACS). We investigated epigenetic regulation of the endothelin-1 (ET-1) gene in PVEC of 1-day and 6-week IUGR rats, and response of IUGR rats to hypoxia.

Results

The maternal nutrient restriction increased the histone acetylation and hypoxia inducible factor-1α (HIF-1α) binding levels in the ET-1 gene promoter of PVEC in IUGR newborn rats, and continued up to 6 weeks after birth. These epigenetic changes could result in an IUGR rat being highly sensitive to hypoxia later in life, causing more significant PAH or pulmonary vascular remodeling.

Conclusions

These findings suggest that epigenetics is closely associated with the development of hypoxic PAH following IUGR, further providing a new insight for improved prevention and treatment of IUGR-related PAH.     

Reciprocal regulation of syndecan-2 and Notch signaling in vascular smooth muscle cells

Vascular cell interactions mediated through cell surface receptors play a critical role in the assembly and maintenance of blood vessels. These signaling interactions transmit important information that alters cell function through changes in protein dynamics and gene expression. Here, we identify syndecan-2 (SDC2) as a gene whose expression is induced in smooth muscle cells upon physical contact with endothelial cells. Syndecan-2 is a heparan sulfate proteoglycan that is known to be important for developmental processes, including angiogenesis. Our results show that endothelial cells induce mRNA expression of syndecan-2 in smooth muscle cells by activating Notch receptor signaling. Both NOTCH2 and NOTCH3 contribute to the increased expression of syndecan-2 and are themselves sufficient to promote its expression independent of endothelial cells. Syndecan family members serve as coreceptors for signaling molecules, and interestingly, our data show that syndecan-2 regulates Notch signaling and physically interacts with NOTCH3. Notch activity is attenuated in smooth muscle cells made deficient in syndecan-2, and this specifically prevents expression of the differentiation marker smooth muscle α-actin. These results show a novel mechanism in which Notch receptors control their own activity by inducing the expression of syndecan-2, which then acts to propagate Notch signaling by direct receptor interaction.     

The outcome of pulmonary hypertension and its association with pulmonary artery dilatation

Background

Pulmonary artery (PA) dilatation is often seen in pulmonary hypertension (PH) and is considered a long-term consequence of elevated pressure. The PA dilates over time and therefore may reflect disease severity and duration. Survival is related to the stage of the disease at the time of diagnosis and therefore PA diameter might be used to predict prognosis. This study evaluates the outcome of patients with pulmonary arterial hypertension (PAH) and chronic thrombo-embolic pulmonary hypertension (CTEPH) and investigates whether PA diameter at the time of diagnosis is associated with mortality.

Methods

Patients visiting an outpatient clinic of a tertiary centre between 2004 and 2018 with a cardiac catheterisation confirmed diagnosis of PAH or CTEPH and a CT scan available for PA diameter measurement were included. PA diameter and established predictors of survival were collected (New York Heart Association (NYHA) class, N‑terminal pro-brain natriuretic peptide (NT-proBNP) level and 6‑min walking distance (6MWD)).

Results

In total 217 patients were included (69% female, 71% NYHA class ≥III). During a median follow-up of 50 (22–92) months, 54% of the patients died. Overall survival was 87% at 1 year, 70% at 3 years and 58% at 5 years. The mean PA diameter was 34.2 ± 6.2 mm and was not significantly different among all the diagnosis groups. We found a weak correlation between PA diameter and mean PA pressure ( r = 0.23, p < 0.001). Male sex, higher age, shorter 6MWD and higher NT-proBNP level were independently associated with mortality, but PA diameter was not.

Conclusion

The prognosis of PAH and CTEPH is still poor. Known predictors of survival were confirmed, but PA diameter at diagnosis was not associated with survival in PAH or CTEPH patients.