3D Imaging Reveals Complex Microvascular Remodeling in the Right Ventricle in Pulmonary Hypertension.

BACKGROUND: Pathogenic concepts of right ventricular (RV) failure in pulmonary arterial hypertension focus on a critical loss of microvasculature. However, the methods underpinning prior studies did not take into account the 3-dimensional (3D) aspects of cardiac tissue, making accurate quantification difficult. We applied deep-tissue imaging to the pressure-overloaded RV to uncover the 3D properties of the microvascular network and determine whether deficient microvascular adaptation contributes to RV failure. METHODS: Heart sections measuring 250-µm-thick were obtained from mice after pulmonary artery banding (PAB) or debanding PAB surgery and properties of the RV microvascular network were assessed using 3D imaging and quantification. Human heart tissues harvested at the time of transplantation from pulmonary arterial hypertension cases were compared with tissues from control cases with normal RV function. RESULTS: Longitudinal 3D assessment of PAB mouse hearts uncovered complex microvascular remodeling characterized by tortuous, shorter, thicker, highly branched vessels, and overall preserved microvascular density. This remodeling process was reversible in debanding PAB mice in which the RV function recovers over time. The remodeled microvasculature tightly wrapped around the hypertrophied cardiomyocytes to maintain a stable contact surface to cardiomyocytes as an adaptation to RV pressure overload, even in end-stage RV failure. However, microvasculature-cardiomyocyte contact was impaired in areas with interstitial fibrosis where cardiomyocytes displayed signs of hypoxia. Similar to PAB animals, microvascular density in the RV was preserved in patients with end-stage pulmonary arterial hypertension, and microvascular architectural changes appeared to vary by etiology, with patients with pulmonary veno-occlusive disease displaying a lack of microvascular complexity with uniformly short segments. CONCLUSIONS: 3D deep tissue imaging of the failing RV in PAB mice, pulmonary hypertension rats, and patients with pulmonary arterial hypertension reveals complex microvascular changes to preserve the microvascular density and maintain a stable microvascular-cardiomyocyte contact. Our studies provide a novel framework to understand microvascular adaptation in the pressure-overloaded RV that focuses on cell-cell interaction and goes beyond the concept of capillary rarefaction.

Rat microbial biogeography and age-dependent lactic acid bacteria in healthy lungs.

The laboratory rat emerges as a useful tool for studying the interaction between the host and its microbiome. To advance principles relevant to the human microbiome, we systematically investigated and defined the multitissue microbial biogeography of healthy Fischer 344 rats across their lifespan. Microbial community profiling data were extracted and integrated with host transcriptomic data from the Sequencing Quality Control consortium. Unsupervised machine learning, correlation, taxonomic diversity and abundance analyses were performed to determine and characterize the rat microbial biogeography and identify four intertissue microbial heterogeneity patterns (P1-P4). We found that the 11 body habitats harbored a greater diversity of microbes than previously suspected. Lactic acid bacteria (LAB) abundance progressively declined in lungs from breastfed newborn to adolescence/adult, and was below detectable levels in elderly rats. Bioinformatics analyses indicate that the abundance of LAB may be modulated by the lung-immune axis. The presence and levels of LAB in lungs were further evaluated by PCR in two validation datasets. The lung, testes, thymus, kidney, adrenal and muscle niches were found to have age-dependent alterations in microbial abundance. The 357 microbial signatures were positively correlated with host genes in cell proliferation (P1), DNA damage repair (P2) and DNA transcription (P3). Our study established a link between the metabolic properties of LAB with lung microbiota maturation and development. Breastfeeding and environmental exposure influence microbiome composition and host health and longevity. The inferred rat microbial biogeography and pattern-specific microbial signatures could be useful for microbiome therapeutic approaches to human health and life quality enhancement.

Rat microbial biogeography and age-dependent lactic acid bacteria in healthy lungs.

The laboratory rat emerges as a useful tool for studying the interaction between the host and its microbiome. To advance principles relevant to the human microbiome, we systematically investigated and defined a multi-tissue full lifespan microbial biogeography for healthy Fischer 344 rats. Microbial community profiling data was extracted and integrated with host transcriptomic data from the Sequencing Quality Control (SEQC) consortium. Unsupervised machine learning, Spearman's correlation, taxonomic diversity, and abundance analyses were performed to determine and characterize the rat microbial biogeography and the identification of four inter-tissue microbial heterogeneity patterns (P1-P4). The 11 body habitats harbor a greater diversity of microbes than previously suspected. Lactic acid bacteria (LAB) abundances progressively declined in lungs from breastfeed newborn to adolescence/adult and was below detectable levels in elderly rats. LAB's presence and levels in lungs were further evaluated by PCR in the two validation datasets. The lung, testes, thymus, kidney, adrenal, and muscle niches were found to have age-dependent alterations in microbial abundance. P1 is dominated by lung samples. P2 contains the largest sample size and is enriched for environmental species. Liver and muscle samples were mostly classified into P3. Archaea species were exclusively enriched in P4. The 357 pattern-specific microbial signatures were positively correlated with host genes in cell migration and proliferation (P1), DNA damage repair and synaptic transmissions (P2), as well as DNA transcription and cell cycle in P3. Our study established a link between metabolic properties of LAB with lung microbiota maturation and development. Breastfeeding and environmental exposure influence microbiome composition and host health and longevity. The inferred rat microbial biogeography and pattern-specific microbial signatures would be useful for microbiome therapeutic approaches to human health and good quality of life.

Repetitive schistosoma exposure causes perivascular lung fibrosis and persistent pulmonary hypertension.

BACKGROUND: Pulmonary hypertension (PH) can occur as a complication of schistosomiasis. In humans, schistosomiasis-PH persists despite antihelminthic therapy and parasite eradication. We hypothesized that persistent disease arises as a consequence of exposure repetition. METHODS: Following intraperitoneal sensitization, mice were experimentally exposed to Schistosoma eggs by intravenous injection, either once or three times repeatedly. The phenotype was characterized by right heart catheterization and tissue analysis. RESULTS: Following intraperitoneal sensitization, a single intravenous Schistosoma egg exposure resulted in a PH phenotype that peaked at 7-14 days, followed by spontaneous resolution. Three sequential exposures resulted in a persistent PH phenotype. Inflammatory cytokines were not significantly different between mice exposed to one or three egg doses, but there was an increase in perivascular fibrosis in those who received three egg doses. Significant perivascular fibrosis was also observed in autopsy specimens from patients who died of this condition. CONCLUSIONS: Repeatedly exposing mice to schistosomiasis causes a persistent PH phenotype, accompanied by perivascular fibrosis. Perivascular fibrosis may contribute to the persistent schistosomiasis-PH observed in humans with this disease.

A Notch3-Marked Subpopulation of Vascular Smooth Muscle Cells Is the Cell of Origin for Occlusive Pulmonary Vascular Lesions.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a fatal disease characterized by profound vascular remodeling in which pulmonary arteries narrow because of medial thickening and occlusion by neointimal lesions, resulting in elevated pulmonary vascular resistance and right heart failure. Therapies targeting the neointima would represent a significant advance in PAH treatment; however, our understanding of the cellular events driving neointima formation, and the molecular pathways that control them, remains limited. METHODS: We comprehensively map the stepwise remodeling of pulmonary arteries in a robust, chronic inflammatory mouse model of pulmonary hypertension. This model demonstrates pathological features of the human disease, including increased right ventricular pressures, medial thickening, neointimal lesion formation, elastin breakdown, increased anastomosis within the bronchial circulation, and perivascular inflammation. Using genetic lineage tracing, clonal analysis, multiplexed in situ hybridization, immunostaining, deep confocal imaging, and staged pharmacological inhibition, we define the cell behaviors underlying each stage of vascular remodeling and identify a pathway required for neointima formation. RESULTS: Neointima arises from smooth muscle cells (SMCs) and not endothelium. Medial SMCs proliferate broadly to thicken the media, after which a small number of SMCs are selected to establish the neointima. These neointimal founder cells subsequently undergoing massive clonal expansion to form occlusive neointimal lesions. The normal pulmonary artery SMC population is heterogeneous, and we identify a Notch3-marked minority subset of SMCs as the major neointimal cell of origin. Notch signaling is specifically required for the selection of neointimal founder cells, and Notch inhibition significantly improves pulmonary artery pressure in animals with pulmonary hypertension. CONCLUSIONS: This work describes the first nongenetically driven murine model of pulmonary hypertension (PH) that generates robust and diffuse occlusive neointimal lesions across the pulmonary vascular bed and does so in a stereotyped timeframe. We uncover distinct cellular and molecular mechanisms underlying medial thickening and neointima formation and highlight novel transcriptional, behavioral, and pathogenic heterogeneity within pulmonary artery SMCs. In this model, inflammation is sufficient to generate characteristic vascular pathologies and physiological measures of human PAH. We hope that identifying the molecular cues regulating each stage of vascular remodeling will open new avenues for therapeutic advancements in the treatment of PAH.

Advances in Proximity Ligation in situ Hybridization (PLISH).

Understanding tissues in the context of development, maintenance and disease requires determining the molecular profiles of individual cells within their native in vivo spatial context. We developed a Proximity Ligation in situ Hybridization technology (PLISH) that enables quantitative measurement of single cell gene expression in intact tissues, which we have now updated. By recording spatial information for every profiled cell, PLISH enables retrospective mapping of distinct cell classes and inference of their in vivo interactions. PLISH has high sensitivity, specificity and signal to noise ratio. It is also rapid, scalable, and does not require expertise in molecular biology so it can be easily adopted by basic and clinical researchers.

Drug repositioning in pulmonary arterial hypertension: challenges and opportunities.

Despite many advances in medical therapy for pulmonary arterial hypertension (PAH) over the past 20 years, long-term survival is still poor. Novel therapies which target the underlying pathology of PAH and which could be added to current vasodilatory therapies to halt disease progression and potentially reverse pulmonary vascular remodeling are highly sought after. Given the high attrition rates, substantial costs, and slow pace of new drug development, repositioning of "old" drugs is increasingly becoming an attractive path to identify novel treatment options, especially for a rare disease such as PAH. We here summarize the limitations of current PAH therapy, the general concept of repurposing and repositioning, success stories of approved repositioned drugs in PAH as well as novel repositioned drugs that show promise in preclinical models of pulmonary hypertension (PH) and are currently tested in clinical trials. We furthermore discuss various data-driven as well as experimental approaches currently used to identify repurposed drug candidates and review challenges for the "repositioning community" with regards to funding and patent and regulatory considerations, and to illustrate opportunities for collaborative solutions for drug repositioning relevant to PAH.

FHIT, a Novel Modifier Gene in Pulmonary Arterial Hypertension.

RATIONALE: Pulmonary arterial hypertension (PAH) is characterized by progressive narrowing of pulmonary arteries, resulting in right heart failure and death. BMPR2 (bone morphogenetic protein receptor type 2) mutations account for most familial PAH forms whereas reduced BMPR2 is present in many idiopathic PAH forms, suggesting dysfunctional BMPR2 signaling to be a key feature of PAH. Modulating BMPR2 signaling is therapeutically promising, yet how BMPR2 is downregulated in PAH is unclear. OBJECTIVES: We intended to identify and pharmaceutically target BMPR2 modifier genes to improve PAH. METHODS: We combined siRNA high-throughput screening of >20,000 genes with a multicohort analysis of publicly available PAH RNA expression data to identify clinically relevant BMPR2 modifiers. After confirming gene dysregulation in tissue from patients with PAH, we determined the functional roles of BMPR2 modifiers in vitro and tested the repurposed drug enzastaurin for its propensity to improve experimental pulmonary hypertension (PH). MEASUREMENTS AND MAIN RESULTS: We discovered FHIT (fragile histidine triad) as a novel BMPR2 modifier. BMPR2 and FHIT expression were reduced in patients with PAH. FHIT reductions were associated with endothelial and smooth muscle cell dysfunction, rescued by enzastaurin through a dual mechanism: upregulation of FHIT as well as miR17-5 repression. Fhit-/- mice had exaggerated hypoxic PH and failed to recover in normoxia. Enzastaurin reversed PH in the Sugen5416/hypoxia/normoxia rat model, by improving right ventricular systolic pressure, right ventricular hypertrophy, cardiac fibrosis, and vascular remodeling. CONCLUSIONS: This study highlights the importance of the novel BMPR2 modifier FHIT in PH and the clinical value of the repurposed drug enzastaurin as a potential novel therapeutic strategy to improve PAH.

Consequences of BMPR2 Deficiency in the Pulmonary Vasculature and Beyond: Contributions to Pulmonary Arterial Hypertension.

Since its association with familial pulmonary arterial hypertension (PAH) in 2000, Bone Morphogenetic Protein Receptor II (BMPR2) and its related signaling pathway have become recognized as a key regulator of pulmonary vascular homeostasis. Herein, we define BMPR2 deficiency as either an inactivation of the receptor, decreased receptor expression, or an impairment of the receptor's downstream signaling pathway. Although traditionally the phenotypic consequences of BMPR2 deficiency in PAH have been thought to be limited to the pulmonary vasculature, there is evidence that abnormalities in BMPR2 signaling may have consequences in many other organ systems and cellular compartments. Revisiting how BMPR2 functions throughout health and disease in cells and organs beyond the lung vasculature may provide insight into the contribution of these organ systems to PAH pathogenesis as well as the potential systemic manifestation of PAH. Here we review our knowledge of the consequences of BMPR2 deficiency across multiple organ systems.

Use of adenosine diphosphate receptor inhibitor prior to left ventricular assist device implantation is not associated with increased bleeding.

Current guidelines recommend adenosine diphosphate receptor inhibitors (ADPRi) be discontinued 5-7 days prior to cardiac surgery due to increased bleeding events, rates of re-exploration, and transfusions. However, the risks of left ventricular assist device (LVAD) implantation in patients taking an ADPRi have not previously been studied. We retrospectively identified 134 eligible patients with ischemic cardiomyopathy that underwent LVAD implantation between July 2009 and August 2013. The cohorts received an ADPRi ≤5 days of surgery (n = 25) versus >5 days prior or not at all (n = 109). Subgroup analyses adjusted for differences in frequency of redo sternotomy between cohorts, excluded patients that received an ADPRi >1 year prior to surgery, and excluded patients with a redo sternotomy. The ADPRi and control groups did not have significant differences in the primary outcomes, intraoperative PRBC units transfused (3.0 vs. 4.0, p = 0.12) or chest tube output within 24 h of surgery (1.66 L vs. 1.80 L, p = 0.61). After adjusting for differences in frequency of redo sternotomy (ADPRi vs. control, 12 vs. 52%, p ≤ 0.001), no significant difference in PRBC units transfused (3.1 vs. 3.5, p = 0.59) or chest tube output (2.04 L vs. 2.04 L, p = 0.98) was seen. No significant difference in 30-day mortality (8.0 vs. 11.0%, p = 0.63), 90-day mortality (16.4 vs. 23.3%, p = 0.42), or length of stay (29.0 vs. 28.0, p = 0.61) was seen. In this single-center experience, use of an ADPRi ≤5 days prior to LVAD implantation was not associated with increased bleeding, length of stay, or mortality.

Pneumonia Pathogen Characterization Is an Independent Determinant of Hospital Readmission.

BACKGROUND: Hospital readmissions for pneumonia occur often and are difficult to predict. For fiscal year 2013, the Centers for Medicare & Medicaid Services readmission penalties have been applied to acute myocardial infarction, heart failure, and pneumonia. However, the overall impact of pneumonia pathogen characterization on hospital readmission is undefined. METHODS: This was a retrospective 6-year cohort study (August 2007 to September 2013). RESULTS: We evaluated 9,624 patients with a discharge diagnosis of pneumonia. Among these patients, 4,432 (46.1%) were classified as having culture-negative pneumonia, 1,940 (20.2%) as having pneumonia caused by antibiotic-susceptible bacteria, 2,991 (31.1%) as having pneumonia caused by potentially antibiotic-resistant bacteria, and 261 (2.7%) as having viral pneumonia. The 90-day hospital readmission rate for survivors (n = 7,637, 79.4%) was greatest for patients with pneumonia attributed to potentially antibiotic-resistant bacteria (11.4%) followed by viral pneumonia (8.3%), pneumonia attributed to antibiotic-susceptible bacteria (6.6%), and culture-negative pneumonia (5.8%) (P < .001). Multiple logistic regression analysis identified pneumonia attributed to potentially antibiotic-resistant bacteria to be independently associated with 90-day readmission (OR, 1.75; 95% CI, 1.56-1.97; P < .001). Other independent predictors of 90-day readmission were Charlson comorbidity score > 4, cirrhosis, and chronic kidney disease. Culture-negative pneumonia was independently associated with lower risk for 90-day readmission. CONCLUSIONS: Readmission after hospitalization for pneumonia is relatively common and is related to pneumonia pathogen characterization. Pneumonia attributed to potentially antibiotic-resistant bacteria is associated with an increased risk for 90-day readmission, whereas culture-negative pneumonia is associated with lower risk for 90-day readmission.

Systolic blood pressure on discharge after left ventricular assist device insertion is associated with subsequent stroke.

BACKGROUND: Stroke is a significant complication in patients supported with continuous-flow left ventricular assist devices (CF-LVAD) and hypertension is a significant risk factor for stroke, but the association between blood pressure and stroke in LVAD patients is not well characterized. METHODS: We identified 275 consecutive patients who survived implant hospitalization between January 2005 and April 2013. Patients were grouped according to systolic blood pressure (SBP) as above a median and below a median of 100 mm Hg by their averaged systolic blood pressure during the 48 hours before discharge from implantation hospitalization. The groups were compared for the primary outcome of time to stroke. RESULTS: The above-median SBP group had mean SBP of 110 mm Hg and the below-median SBP group had mean SBP of 95 mm Hg. There were no significant between-group differences in body mass index, smoking, vascular disease, hypertension, atrial fibrillation, or prior stroke. During a mean follow-up of 16 months, stroke occurred in 16% of the above-median SBP group vs in 7% of the below-median SBP group (hazard ratio, 2.38; 95% confidence interval, 1.11-5.11), with a similar proportion of hemorrhagic and ischemic strokes in each group. In Cox proportional hazard models adjusting for age, diabetes, or prior stroke, the hazard ratio remained statistically significant. SBP as a continuous variable predictor of stroke had an area under the curve of 0.64 in a receiver operating characteristic curve analysis. CONCLUSIONS: In this large, CF-LVAD cohort, elevated SBP was independently associated with a greater risk of subsequent stroke. These results identify management of hypertension as a potential modifiable risk factor for reducing the incidence of stroke in patients supported by CF-LVAD.