Centralized and cross-referenced biobanking resource in PH with high-throughput molecular profiling
Network and mathematical analytics in PH
Despite the growing prevalence of pulmonary hypertension (PH), therapeutic advances have been slowed by little financial support and small numbers of dedicated researchers. Particularly in countries with limited medical care, patients are in desperate need of medications that prevent or reverse PH. We are uniquely positioned to accelerate systems-wide discovery and therapeutic strategy for PH — by identifying the elusive molecular origins of PH, their roles within interconnected molecular processes, and the response of those connections to pharmacologic interventions.
To do so, we are combining in silico network-based theory with biological experimentation of PH, as the Chan Lab has described for studies of microRNAs (A) 4, 6, 8. The combination of such disparate disciplines in complex human disease has rarely been attempted but will have transformative implications. Specifically, in contrast to traditional reductionist approaches, our prior success has demonstrated that this methodology is more effective in deciphering the in vivo mechanisms of synergy and redundancy by which cohorts of molecules modulate target genes and control resultant PH. Illustrated by our prior findings, we are poised to apply a molecular systems perspective to develop new tools to diagnose PH, identify new conditions that predispose to PH, and define new patient cohorts at risk for PH. We are also among the few, if not only, centers with rigorous cross-disciplinary expertise to initiate “network pharmacology” screens to identify new therapeutics computationally for human PH. Perhaps most importantly, our studies hold great promise for application in other diseases beyond PH for rapid identification of master molecular regulators hidden in the architecture of their gene networks.
Stephen Y. Chan, MD, PhD | Bio
- Chan SY, Zhang YY, Hemann C, Mahoney CE, Zweier JL and Loscalzo J. MicroRNA-210 controls mitochondrial metabolism during hypoxia by repressing the iron-sulfur cluster assembly proteins ISCU1/2. Cell Metabolism. 2009;10:273-84.
- White K, Lu Y, Annis S, Hale AE, Chau BN, Dahlman JE, Hemann C, Opotowsky AR, Vargas SO, Rosas I, Perrella MA, Osorio JC, Haley KJ, Graham BB, Kumar R, Saggar R, Saggar R, Wallace WD, Ross DJ, Khan OF, Bader A, Gochuico BR, Matar M, Polach K, Johannessen NM, Prosser HM, Anderson DG, Langer R, Zweier JL, Bindoff LA, Systrom D, Waxman AB, Jin RC and Chan SY. Genetic and hypoxic alterations of the microRNA-210-ISCU1/2 axis promote iron-sulfur deficiency and pulmonary hypertension. EMBO molecular medicine. 2015;7:695-713.
- Bertero T, Cotrill KA, Lu Y, Haeger CM, Dieffenbach P, Annis S, Hale A, Bhat B, Kaimal V, Zhang YY, Graham BB, Kumar R, Saggar R, Saggar R, Wallace WD, Ross DJ, Black SM, Fratz S, Fineman JR, Vargas SO, Haley KJ, Waxman AB, Chau BN, Fredenburgh LE and Chan SY. Matrix remodeling promotes pulmonary hypertension through feedback mechanoactivation of the YAP/TAZ-miR-130/301 circuit Cell Reports. 2015;13:1016-1032.
- Bertero T, Cottrill K, Krauszman A, Lu Y, Annis S, Hale A, Bhat B, Waxman AB, Chau BN, Kuebler WM and Chan SY. The microRNA-130/301 family controls vasoconstriction in pulmonary hypertension. J Biol Chem. 2014;290:2069-2085.
- Bertero T, Cottrill KA, Annis S, Bhat B, Gochuico BR, Osorio JC, Rosas I, Haley KJ, Corey KE, Chung RT, Nelson Chau B and Chan SY. A YAP/TAZ-miR-130/301 molecular circuit exerts systems-level control of fibrosis in a network of human diseases and physiologic conditions. Scientific reports. 2015;5:18277.
- Bertero T, Lu Y, Annis S, Hale A, Bhat B, Saggar R, Saggar R, Wallace WD, Ross DJ, Vargas SO, Graham BB, Kumar R, Black SM, Fratz S, Fineman JR, West JD, Haley KJ, Waxman AB, Chau BN, Cottrill KA and Chan SY. Systems-level regulation of microRNA networks by miR-130/301 promotes pulmonary hypertension. J Clin Invest. 2014;124:3514-28.
- Bertero T, Oldham WM, Cottrill KA, Pisano S, Vanderpool RR, Yu Q, Zhao J, Tai Y, Tang Y, Zhang YY, Rehman S, Sugahara M, Qi Z, Gorcsan J, 3rd, Vargas SO, Saggar R, Saggar R, Wallace WD, Ross DJ, Haley KJ, Waxman AB, Parikh VN, De Marco T, Hsue PY, Morris A, Simon MA, Norris KA, Gaggioli C, Loscalzo J, Fessel J and Chan SY. Vascular stiffness mechanoactivates YAP/TAZ-dependent glutaminolysis to drive pulmonary hypertension. J Clin Invest. 2016;126:3313-35.
- Parikh VN, Jin RC, Rabello S, Gulbahce N, White K, Hale A, Cottrill KA, Shaik RS, Waxman AB, Zhang YY, Maron BA, Hartner JC, Fujiwara Y, Orkin SH, Haley KJ, Barabasi AL, Loscalzo J and Chan SY. MicroRNA-21 integrates pathogenic signaling to control pulmonary hypertension: results of a network bioinformatics approach. Circulation. 2012;125:1520-32.
Biobanking of PH blood/plasma
The Translational Research Core Laboratory and Biobank of VMI is responsible for the biobanking of PH blood samples.
- Whole blood is transported to the Core Lab and processed for serum, plasma, DNA, buffy coat, PBMC, etc.
- All samples are bar-coded and inventoried using the Freezerworks database.
- The Freezerworks database is linked to the clinical database maintained by the clinical team.
- Samples are distributed to research teams in a timely fashion.
- The Core Lab is the central hub that triages blood sample distribution to maximize the yield of each blood sample.
Biobanking of PH tissue/cells: PAH-PAECs and PAH-PASMCs
- Isolation, characterization, and biobanking of pulmonary vascular smooth muscle cells, endothelial cells, and adventitial fibroblasts from patients with pulmonary arterial hypertension (PAH) and non-PAH lungs
- Providing VMI researchers with primary human cell cultures for PAH- and pulmonary vascular biology-related studies
- Help with experimental techniques specifically adjusted to primary human cells
Elena Goncharova, PhD | Bio
Novel rodent models of PH (HFpEF-PH)
- Our group has recently developed a two-hit model of PH-HFpEF, which combines endothelial injury in rats with multiple features of metabolic syndrome (Obese ZSF1/SU5416).
- Based on the hemodynamic evaluation of 36 mouse strains exposed to standard and high fat diet (HFD), we identified and validated a HFD-AKR/J mouse model of PH-HFpEF.
- All clinical features seen in PH-HFpEF patients, including elevated LV end-diastolic pressure and RV systolic pressure, preserved LV ejection fraction, and biventricular hypertrophy, are consistently observed in our models (Figure 3 and 4).
This is significant because PH-HFpEF is the most frequent cause of pulmonary hypertension and to date has not been identified an effective treatment. A mouse model will help to understand the pathogenesis of the disease and to identify novel therapeutic targets.
Ana Mora, MD | Bio
Ex-vivo human lung physiology
Dr. Rojas’ laboratory is using novel approaches to increase the number of transplant acceptable lungs in an attempt to lessen wait-list times and improve outcomes post-transplant. Using a recently developed technique called Ex Vivo Lung Perfusion (EVLP), our group uses lungs deemed unsuitable for transplant and attempts to improve their quality by various approaches such as stem cell therapy and newly developed drugs. In this manner, we hope to increase the number of lungs that can be used in the clinical setting for transplant in patients with pulmonary hypertension, as well as many other end-stage lung diseases.
Mauricio Rojas, MD | Bio
Single cell high-throughput RNA-Seq
- We have discovered a series of biomarkers of pulmonary arterial hypertension associated with systemic sclerosis (SSc-PAH).
- We have identified a series of mRNA markers expressed by peripheral blood mononuclear cells of patients with SSc-PAH
- We have identified a series of serum proteins that are elevated in patients with SSc-PAH.
This is significant because it will permit earlier recognition and diagnosis.
Robert Lafyatis, MD | Bio
VMI Calculator measures the wall thickness of small pulmonary arteries
Automated Measurement of Blood Vessels in Tissues from Microscopy Images. Kelly NJ, Dandachi N, Goncharov DA, Pena AZ, Radder JE, Gregory AD, Lai YC, Leme AS, Gladwin MT, Goncharova EA, St Croix CM, Shapiro SD. Curr Protoc Cytom. 2016 Oct 10;78:12.44.1-12.44.13. doi: 10.1002/cpcy.10. [PMID 27723088]
The quantification of tunica media thickness in histological cross sections is a ubiquitous exercise in cardiopulmonary research, yet the methods for quantifying medial wall thickness have never been rigorously examined with modern image analysis tools. As a result, inaccurate and cumbersome manual measurements of discrete wall regions along the vessel periphery have become common practice for wall thickness quantification. The aim of this study is to introduce, validate, and facilitate the use of an improved method for medial wall thickness quantification. We describe a novel method of wall thickness calculation based on image skeletonization and compare its results to those of common techniques. Using both theoretical and empirical approaches, we demonstrate the accuracy and superiority of the skeleton-based method for measuring wall thickness while discussing its interpretation and limitations. Finally, we present a new freely available software tool, the VMI Calculator, to facilitate wall thickness measurements using our novel method.
Access the software here: http://www.vmi.pitt.edu/resources/VMIcalculator.html
Neil Kelly, PhD | Bio