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Berk Lab

URMC / Cardiovascular Research Institute / Research / Berk Lab / Research Projects

Research Projects

The three major projects currently active in my laboratory are:

1. The genetic basis for vascular intima formation, vascular remodeling and inflammation: PDE10A, PNPT1, and RPL17.

A long-term goal in treating atherosclerosis and hypertension is to understand the mechanisms that regulate the structure of blood vessels; a process termed “vascular remodeling.” An important predictive phenotype for human cardiovascular disease (CVD), is vascular remodeling in the carotid artery, determined by the measurement termed intima-media thickening (IMT). IMT is mediated by EC dysfunction, vascular smooth muscle cell (VSMC) growth, as well as inflammatory cell accumulation and activation. These pathological processes are stimulated by a disturbed flow pattern (d-flow), while being minimized by steady (s-flow). To identify genes that contribute to IMT we used partial ligation of the left carotid to create d-flow, resulting in intima formation that models human carotid IMT. Among 17 inbred mouse strains we found strain-dependent intima formation (lowest in C3HeB/FeJ (C3H/F) and greatest in SJL/J (SJL). We performed a quantitative trail locus (QTL) analysis of vascular remodeling alleles in backcrosses of C3H/F and SJL mouse strains and identified three QTLs, termed Intima modifier loci (lm1 on chromosome 2, lm2 on chromosome 11 and Im3 on chromosome 18), that regulate intimal thickening. Intima in SJL carotids was associated with increased inflammation (CD45+ staining) vs. C3H/F. Using a combination of analyses including congenic fine mapping, genome-wide association with both SNPs and the Hybrid Mouse Diversity Panel (HMDP), RNA sequencing, and biological pathway analysis we identified at least 12 candidate intima and inflammation genes. Based on expression studies, putative function, and the analysis above, we validated several genes of which the following remain the focus of the Berk lab; PDE10A, PNPT1, and RPL17.

  1. Korshunov, VA, Berk BC.  Genetic modifier loci linked to intima formation induced by low flow in the mouse carotid.  Arterioscler Thromb Vasc Biol; 2009, 29:47-53. PMID: 18948632
  2. Smolock EM, Burke RM, Wang C, Thomas T, Batchu SN, Qiu X, Zettel M, Fujiwara K, Berk BC, Korshunov VA.  Intima modifier locus 2 controls endothelial cell activation and vascular permeability.  Physiol Genomics; 2014, 46:624-33. PMID: 24986958
  3. Simons M, Alitalo K, Annex BH, Augustin HG, Beam C, Berk BC, Byzova T, Carmeliet P, Chilian W, Cooke JP, Davis GE, Eichmann A, Iruela-Arispe ML, Keshet E, Sinusas AJ, Ruhrberg C, Woo YJ, Dimmeler S. State-of-the-Art Methods for Evaluation of Angiogenesis and Tissue Vascularization A Scientific Statement From the American Heart Association. Circ. Res.; 2015, 116:E99-E132. PMID: 25931450
  4. Heo KS, Berk BC, Abe JI. Disturbed flow-induced endothelial proatherogenic signaling via regulating post-translational modifications and epigenetic events. Antioxid Redox Signal; 2016, 25:435-450. PMID: 26714841
  5. Korshunov VA, Smolock EM, Wines-Samuelson ME, Faiyaz A, Mickelsen DM, Quinn B, Pan C, Dugbartey GJ, Yan C, Doyley MM, Lusis AJ, Berk BC. Natriuretic Peptide Receptor 2 Locus Contributes to Carotid Remodeling. J Am Heart Assoc. 2020, 9: In Press. PMID: 32394795.

A. PDE10A Regulation and Function in CVD.

Vascular injuryIn the analysis above, we identified cyclic nucleotide phosphodiesterase 10A (PDE10A) as a candidate gene for intimal thickening. We found that PDE10A expression was markedly elevated in the intimal VSMC-like cells and macrophages in mouse models of intimal thickening and in human atherosclerotic lesions compared to healthy vessels. Furthermore, in PDE10A knock-out mice or mice treated with TP-10 (specific PDE10A inhibitor), VSMC growth and macrophage inflammatory activation were inhibited. Based on biochemical studies in VSMC and macrophages, we will test the model shown to study the mechanisms by which PDE10A contributes to cardiovascular disease (Figure). PDE10A stimulates a synthetic VSMC phenotype transition by inhibiting cAMP-PKA-mediated phosphorylation and stabilization of MYOCD; thus suppressing the VSMC contractile gene program. In macrophages, PDE10A stimulates inflammation by two mechanisms: 1) it inhibits the cAMP-PKA regulation of NF-kB activity, thereby increasing mRNA expression of NLRP3 inflammasome proteins and other inflammasome-independent cytokines, chemokines and adhesion molecules. 2) PDE10A prevents NLRP3 protein degradation. The likely mechanism is that cAMP can bind to NLRP3 (with nucleotide binding domain), which stimulates NLRP3 protein ubiquitination and degradation. This in turn decreases NLRP3 inflammasome-mediated formation of IL-1b and IL-1865. Thus, we propose that PDE10A contributes to vascular occlusive disease by stimulating SMC phenotype transition and macrophage-mediated inflammation (Figure).

Aim 1. Determine the role of PDE10A in intima formation and vascular remodeling after injury using both genetic and pharmacological approaches.

We will use mouse femoral artery injury and vein graft models to examine effects of PDE10A deficiency on intimal formation and graft atherosclerosis. Evaluate pharmacological effects of PDE10A inhibitors on injury-induced intima formation in mice, and spontaneous remodeling of human saphenous vein ex vivo. Determine the role of vascular- and bone marrow-derived cells in PDE10A-mediated regulation of injury-induced intimal hyperplasia.

Aim 2. Define the mechanisms for PDE10A regulation of vascular pathology: regulation of PDE10A expression, transition of SMC phenotype, and stimulation of vascular inflammation.

We will determine the role of PDE10A-cAMP-PKA-GSK3b signaling cascade in regulating myocardin protein stability and SMC contractile gene program in SMCs. Determine the role of PDE10A in macrophage NF-kB signaling and expression of NLRP3 inflammasome proteins and other inflammatory mediators; and in inflammasome activation through inhibiting cAMP-mediated NLRP3 protein ubiquitination and degradation. Because PDE10A is a highly regulated gene in response to environmental stimuli, we will use bioinformatic and genetic approaches to characterize the cis- and trans-regulatory elements important in controlling PDE10A gene expression. We will prove that increased PDE10A expression, by inhibiting cAMP signaling, promotes a synthetic SMC phenotype transition and macrophage inflammasome expression/activation; and thus stimulates intimal hyperplasia. The overall objective is to investigate the mechanisms that regulate expression of PDE10A, and PDE10A’s specific enzymatic role in the processes responsible for intimal hyperplasia. Our studies should yield novel therapeutic strategies to limit pathologic intimal hyperplasia given that drugs for human use that inhibit PDE10A have been clinically approved.

  1. Jeon KI, Xu X, Aizawa T, Lim JH, Jono H, Kwon DS, Abe JI, Berk BC, Li JD, Yan C.  Vinpocetine inhibits NF-{kappa}B-dependent inflammation via an IKK-dependent but PDE-independent mechanism.  Proc Natl Acad Sci; 2010, 107:9795-9800. PMID: 20448200.

B. PNPT1 Regulation and Function in CVD.

Polyribonucleotide nucleotidyltransferase 1 (Pnpt1) is a 3’-5’ exoribonuclease that is required for import and processing of RNA in mitochondria. Pnpt1 expression correlated with decreased intima growth and inflammation in the partial carotid ligation model, suggesting it was protective. The specific goal of this project is to understand how Pnpt1 limits inflammation, vascular remodeling and atherosclerosis, focusing on novel transcriptional programs and mechanisms that link d-flow-mediated signaling through mitochondrial homeostasis, mitophagy/autophagy and cellular RNA processing pathways to EC dysfunction and CVD. While intima growth is primarily due to proliferation of VSMC and fibroblast-like cells, we focus on d-flow-mediated effects on EC because we believe these signals are initiating events. d-flow inhibited Pnpt1 function in EC and Pnpt1 deficiency exacerbated mitochondrial-stress, as measured by mt-ROS generation as well as autophagy.  RNA-Seq analyses of Pnpt1 expression under s- and d-flow identified a novel and significant role for the TFAP2b/c transcription factor to repress genes whose expression is required for normal EC function.  We hypothesize that Pnpt1 is a flow regulated enzyme that is critical to mitochondrial homeostasis and whose function is to inhibit vascular inflammation and intima growth, thereby limiting CVD. We are studying three major aspects of Pnpt1 function as shown in the figure.

Aim1Determine the role of Pnpt1 in the pathogenesis of intima growth and atherosclerosis in vivo. We will compare EC-Pnpt1-KO mice to littermate controls in mouse models of CVD including: 1) partial carotid ligation with d-flow-mediated intima growth, and 2) atherosclerosis lesion formation in ApoE-/- aortic and carotid d-flow models. Determine the flow dependent transcriptional program regulated by Pnpt1. Bioinformatics of gene expression regulated by Pnpt1 identified a significant role for the transcription factor TFAP2b/c. RNA-Seq data analyses, ChIP protocols and knockout mouse models of Tfap2b/c will be used to study the role of TFAP2b/c in regulating EC function in CVD. Study the flow dependent role of Pnpt1 in regulating EC metabolism and mitochondrial function. We will measure mitochondrial RNA processing by qPCR, mitochondrial function by mitochondrial -ROS production, mitochondrial morphology, and mitochondrial -fission and fusion. We will determine the effects of Pnpt1 phosphorylation on translation of mitochondrial proteins by measuring expression of several key mitochondrial proteins.

  1. Smolock EM, Machleder DE, Korshunov VA, Berk BC.  Identification of a genetic locus on chromosome 11 that regulates leukocyte infiltration in mouse carotid artery.  Arterioscler Thromb Vas Biol; 2013, 33(5):1014-9. PMID: 23448970

C. RpL17 (Ribosomal protein 17) mediates vascular endothelial dysfunction and intima formation. Similar to PNPT1, KEGG analysis, microarray, and chromosome location identified RpL17 as a candidate gene for IMT. Specifically, decreased expression of RpL17 correlated with increased IMT [6, 12]. RpL17 is crucial for proper cleavage of the 5.8S and 28S ribosomal RNAs, making it essential for formation of mature ribosomes. It is located within the nascent polypeptide exit tunnel of the ribosome, making it vital for high-fidelity processing of newly translated proteins. RpL17 is an essential component of the ribosome, such that complete removal of both alleles causes embryonic lethality in mice and humans. Therefore, all experiments in this project will use heterozygous (RpL17+/-) mice. Experimental results provide a solid rationale for RpL17 as a pathogenic CVD gene. 1) There is 8-fold less RpL17 in SJL carotids compared to C3H/F carotids, with 40-fold greater intima growth in response to d-flow in SJL mice [8]. 2) There are markedly decreased levels of RpL17 in EC of human atherosclerotic carotids compared to healthy controls, as well as in the ApoE-/- mouse model of hyper-lipidemia. 3) Following carotid ligation of EC-restricted (Cdh5-Cre termed EC-RpL17) and ubiquitous (CMV-Cre) conditional heterozygous mice, RpL17+/- mice showed increased intima, inflammation and cell proliferation. 4) RNA-Seq and KEGG pathway analysis of steady-state gene expression in EC-RpL17 vs. RpL17+/+ (Control) mouse lung microvascular EC (MLMEC) revealed significantly decreased gene expression in ribosome-associated, translation, and oxidative phosphorylation (OXPHOS) pathways. 5) Functional consequences of decreased RpL17 in MLMEC and human umbilical vein EC (HUVEC) include increased reactive oxygen species (ROS) and ER stress, decreased OXPHOS and increased glycolysis: metabolic findings consistent with a growth and angiogenic phenotype. 6) There were dramatic and specific changes in translational efficiency of several glycolytic and ROS producing enzymes, most notably PFKFB3 and NOX4. Thus, we hypothesize that RpL17 is an essential ribosomal protein that controls translation of EC proteins and metabolites that inhibit IMT and vascular remodeling. We propose the following Aims (Figure).

Schematic of AimsAim 1: Show that reduced EC RpL17 expression is sufficient to promote IMT and vascular remodeling. We will compare EC-RpL17 mice to Controls in carotid d-flow to study RpL17 effects on inflammation, endothelial to mesenchymal transition, and VSMC growth. Aim 2: Define the role of specific proteins whose translational efficiency is regulated by RpL17 in mediating intima formation and inflammation. Ribogenesis, RpL17 subcellular location, and ribosome profiling followed by mRNA analysis in polysome fractions will be used to define the biological role of RpL17 in vascular remodeling. Aim 3: Determine the RpL17-mediated metabolic changes in EC OXPHOS and glycolysis resulting from altered translational control which contributes to IMT. We will measure translational efficiency of key proteins involved in metabolism (glycolysis, TCA cycle, and OXPHOS), ROS and antioxidants, ER stress, and mitochondrial dysfunction. We will focus on the key proteins involved in energy production, as well as those involved in protein translation and mRNA stability. Using mass spectrometry we will identify secreted proteins and metabolites, and assay their effects on EC and VSMC function. We will establish causality of these factors using inhibitors, siRNA, and knockout mouse models.

  1. Smolock EM, Korshunov VA, Glazko G, Qiu XD, Gerloff J, Berk BC.  Ribosomal protein L17, RpL17 is an inhibitor of vascular smooth muscle growth and carotid intima formation.  Circulation; 2012, 126:2418-2427.  PMID: 23065385
  2. Smolock EM, Machleder DE, Korshunov VA, Berk BC.  Identification of a genetic locus on chromosome 11 that regulates leukocyte infiltration in mouse carotid artery.  Arterioscler Thromb Vas Biol; 2013, 33(5):1014-9.  PMID: 23448970

2The role of Cyclophilin A (CypA) in cardiovascular disease.

CypA is a ubiquitous and highly conserved protein with peptidyl prolyl isomerase activity.  It was first identified as the cytosolic binding partner of the immunosuppressive drug cyclosporin A (CsA) whereupon the complex inhibits the transcription of immune response related genes and prevents proliferation of T cells.

CD147Numerous pioneering studies from my laboratory revealed that CypA contributes to the pathogenesis of vascular disease, including carotid intima-media thickening, atherosclerosis, peripheral artery disease, pulmonary arterial hypertension, platelet activation and blood-brain barrier dysfunction. Oxidative stress and inflammation play key roles in the development of cardiovascular diseases. Increased CypA expression both promotes the generation of cellular oxidative stress by increasing activity of NADPH oxidase but CypA is also secreted in a regulated manner involving RhoA kinase and ROCK in response to oxidative stress. Extracellular CypA (eCypA) modulates cellular functions similar to intracellular CypA such as inflammation and proliferation, but also unique pathways including increasing apoptosis, migration, matrix degradation and generation of Reactive Oxygen Species (ROS).  A putative receptor for eCypA, CD147, also known as Basigin or EMMPRIN, has been demonstrated to play a role in signal transduction although aspects of eCypA signaling remain after CD147 depletion in cells or knockout from mouse models.  Previously it has been difficult to determine the relative contributions of intracellular versus extracellular CypA in disease. However, together with a collaborator we have used a cell impermeable CsA derivative to inhibit eCypA activity and thereby reduce eCypA-mediated EC apoptosis, and VSMC ERK1/2 activation and proliferation both in cultured cells and in a rat model of pulmonary hypertension.

The lab continues to research the mechanism of action of CypA in CVD as well as pursuing the identification of the CypA receptor that initiates signal transduction upon eCypA binding. In CVD we are focusing on the role of CypA in cerebrovascular disease such as stroke, small vessel disease and cognitive dysfunction and the blood brain barrier.   

  1. Bell RD,Winkler EA,Singh I, Sagare AP, Deane R,Wu Z, Holtzman DM, Betscholtz C, Armulik A, Sallstrom J, Berk BC, Zlokovic BV.  Apolipoprotein E controls cerebrovascular integrity via cyclophilin A.  Nature; 2012, 485:512-6.  PMID: 22622580
  2. Perrucci GL, Straino S, Corlianò M, Scopece A, Berk BC, Lombardi F, Pompilio G, Capogrossi MC, Nigro P.  Cyclophilin A modulates bone marrow-derived CD117(+) cells and enhances ischemia-induced angiogenesis via the SDF-1/CXCR4 axis.  Int J Cardiol. 2016, 212:324-35.  PMID: 27057951
  3. Xue, C, Sowden, M. and Berk, BC.  Extracellular cyclophilin A, especially acetylated, causes pulmonary hypertension by stimulating endothelial apoptosis, redox stress and inflammation.  ATVB  2017, 37:1138-1146. PMID: 28450293
  4. Xue, C, Senchanthisai, S, Sowden, M, Pang, J, White, J. Berk, B. Endothelial-to-Mesenchymal Transition and Inflammation Play Key Roles in Cyclophilin A-Induced Pulmonary Arterial Hypertension. Hypertension 2020. In press.

3. Promoting nerve recovery by inhibiting inflammation and promoting its resolution

Recently I started a new area of research focused on the role of inflammation in peripheral nerve and spinal cord injury. This occurred because of three factors. First, I had an accident 11 years ago that caused a spinal cord injury (SCI), which has made me a quadriplegic  Second, the animal models for SCI and peripheral nerve injury became standardized. Third, our studies of inflammation in cardiovascular disease overlapped with likely mechanisms in nerve injury. Importantly, my recent NIH-funded work (with my co-investigator Dr. Chen Yan) that focuses on cyclic nucleotide phosphodiesterase 10A (PDE10A), brought together my CVD and neuroscience interests. In a mouse model of traumatic injury, we found that IL-1β mRNA was dramatically induced (67-fold) on day 3 after sciatic nerve injury (SNI). Recent data suggest that uncontrolled inflammasome activation, which mediates caspase-1 activation and subsequent processing of pro-IL-1β into its mature forms, leads to sustained inflammation and poor functional recovery. We found NLRP3 and its adaptor protein Pycard gene expression were increased in injured tissues.Spinal Cord Injury Furthermore, we found that PDE10A was highly induced by injury. In vitro we showed that PDE10A inhibition with the specific drug TP-10, decreased macrophage IL-1β secretion after stimulation by LPS and nigericin. Our approach to improve tissue recovery will use two strategies; decrease acute inflammation and promote active resolution of inflammation. We found previously that PDE10A was a key mediator of inflammation in injured blood vessels; and in recent data, exhibited similar properties in microglia and macrophages. The overall goal of this project is to test TP-10 as a novel therapeutic strategy in pre-clinical models of SCI. Based on our data and previous literature, we hypothesize that PDE10A inhibition will reduce tissue damage and improve motor function after SCI by decreasing inflammasome activation. Aim 1 To optimize a nanoparticle-mediated TP-10 delivery system for SCI. We will examine the effects of TP-10 treatment using a rat (6 month, C57/Bl6) thoracic (T9) contusion SCI model.  We have established several drug delivery systems focusing on hybrid depots comprised of nanoparticles (for drug loading) embedded within degradable hydrogel to treat musculoskeletal injury. TP-10 is a hydrophobic PDE10 inhibitor, so it will be loaded and released from several different types of nanoparticles (NPs). The major goals of this aim are to find the best TP-10 loading efficiency and loading capacity within NPs. To achieve localized, and slow-release properties, we will use PEG hydrogel for drug depots that will be placed at the injured spinal cord immediately after SCI. To assay degradation rate of locally delivered TP-10, NPs will be labeled with rhodamine, and the intensity change of fluorescence (rhodamine: 553/627) will be measured by IVIS imaging system weekly for 8 weeks. Aim 2: To determine the effects of TP-10/nanoparticle treatment on the restoration of motor function after SCI. The major goals of Aim 2 will be to show at optimal doses of TP-10 based on Aim 1 the change in motor function over time after SCI, and to characterize changes in the inflammatory response (weeks 1, 2, 3, 4, 5 and 7). Briefly, we will deliver TP-10/NPs, or Control NPs immediately after SCI (single dose at 1mg/kg as an example). We will assess motor recovery after SCI using Basso, Beattie and Bresnahan (BBB) and CatWalk gait analysis. We will assay nerve morphology, muscle function, and fiber size. We will measure PDE10A expression in spinal cord by IHC. We will also perform double IHC staining of PDE10A and cell specific markers for macrophages, neurons, astrocytes, and oligodendrocytes. To measure inflammation and its resolution, we will assay cytokine and lipid mediators; at the pro-inflammatory phase (week 1) and the resolution phase (week 5) following injury. We will measure inflammasome components by western blot, levels of cyclic nucleotides, cytokines and vascular inflammatory proteins. We will measure lipids by LC-MS-MS16; focusing on specialized pro resolving mediators (SPMs), such as RvD1, RvD2, Mar1. The project, if successful will characterize SCI inflammation (especially the inflammasome) and its resolution (especially SPMs). We will then relate changes in these parameters of inflammation to neuromuscular function after SCI. If TP–10 is effective at decreasing inflammation and improving neuromuscular function; these results would justify performing studies in larger animals and ultimately nonhuman primates.

  1. Garin G, Abe JI, Mohan A, Lu W, Yan C, Newby AC, Rhaman A, Berk BC. Flow antagonizes TNF-alpha signaling in endothelial cells by inhibiting caspase-dependent PKC-zeta processing.  Circ Res; 2007, 101:97-106. PMID: 17525369
  2. Abe JI, Berk BC. Cezanne paints inflammation by regulating ubiquitination.  Circ Res; 2013, 112(12):1526-8. PMID: 23743222
  3. Abe JI, Berk BC.  Athero-prone flow activation of the SREBP2-NLRP3 Inflammasome mediates focal atherosclerosis.  Circulation; 2013, 128(6):579-82. PMID: 23838164