Discovering and Developing Successful Cardiovascular Therapeutics: A Conversation With James N. Topper, MD, PhD.

Dr James (also known as Jamie) N. Topper, MD, PhD, serves as Managing General Partner at Frazier Healthcare Partners, where he leads the Life Science Venture practice. In 2011, and 2016, he was named to the Midas List of leading venture capitalists, and, in 2013, he was recognized by Forbes as one of the top 10 healthcare investors. He has >25 years of experience working with entrepreneurs to found and build successful therapeutics-focused companies. Dr Topper holds a BS from the University of Michigan. He received an MD and PhD (in biophysics) from Stanford University School of Medicine. He completed postgraduate training in internal medicine and cardiovascular disease at the Brigham and Women's Hospital in Boston and is board certified in both disciplines.

Conditional expression of Smad7 in pancreatic beta cells disrupts TGF-beta signaling and induces reversible diabetes mellitus.

Identification of signaling pathways that maintain and promote adult pancreatic islet functions will accelerate our understanding of organogenesis and improve strategies for treating diseases like diabetes mellitus. Previous work has implicated transforming growth factor-beta (TGF-beta) signaling as an important regulator of pancreatic islet development, but has not established whether this signaling pathway is required for essential islet functions in the adult pancreas. Here we describe a conditional system for expressing Smad7, a potent inhibitor of TGF-beta signaling, to identify distinct roles for this pathway in adult and embryonic beta cells. Smad7 expression in Pdx1+ embryonic pancreas cells resulted in striking embryonic beta cell hypoplasia and neonatal lethality. Conditional expression of Smad7 in adult Pdx1+ cells reduced detectable beta cell expression of MafA, menin, and other factors that regulate beta cell function. Reduced pancreatic insulin content and hypoinsulinemia produced overt diabetes that was fully reversed upon resumption of islet TGF-beta signaling. Thus, our studies reveal that TGF-beta signaling is crucial for establishing and maintaining defining features of mature pancreatic beta cells.

Transforming growth factor-beta receptors localize to caveolae and regulate endothelial nitric oxide synthase in normal human endothelial cells.

Caveolae (sphingolipid- and cholesterol-rich, 100 nm flask-shaped invaginations of the cell membrane) serve as a nexus of cell signalling. In the present study caveolin-rich lipid raft domains were extracted from HUVEC (human umbilical-vein endothelial cells) using both density gradient and immunoprecipitation techniques, and demonstrated localization of the TGF-beta (transforming growth factor-beta) receptors TbetaRI and TbetaRII to the Cav-1 (caveolin-1)-enriched raft fractions of these normal, human endothelial cells. Immunoprecipitation demonstrated an association between TbetaRI and TbetaRII, as well as an association of the TbetaRs receptors with Cav-1 and eNOS (endothelial nitric oxide synthase), suggesting a mutual co-localization to caveolae; after treatment of HUVEC with 5 ng/ml TGF-beta1 for 15 min, however, co-precipitation of eNOS with TbetaRI, TbetaRII and Cav-1 was diminished. The loss of immunoprecipitable eNOS from Cav-1-enriched fractions was accompanied by a decrease both in phosphorylation of eNOS and in enzymatic activity (conversion of arginine into citrulline). No change in the localization of eNOS to morphologically distinct caveolae could be detected by electron microscopy after treatment of HUVEC with TGF-beta1 for 20 min. The results of these investigations provide evidence that TbetaRI interacts with eNOS in the caveolae of normal, human endothelial cells and has a regulatory function on basal eNOS enzymatic activity.

Common genomic response in different mouse models of beta-adrenergic-induced cardiomyopathy.

BACKGROUND: Although beta-adrenergic receptor (AR) blockade therapy is beneficial in the treatment of heart failure, little is known regarding the transcriptional mechanisms underlying this salutary action. METHODS AND RESULTS: In the present study, we screened mice overexpressing Gsalpha, beta1AR, beta2AR, or protein kinase A to test if a common genomic pathway exists in different models with enhanced beta-adrenergic signaling. In mice overexpressing Gsalpha, differentially expressed genes were identified by mRNA profiling. In addition to well-known markers of cardiac hypertrophy (atrial natriuretic factor, CARP, and beta-myosin heavy chain), uncoupling protein 2 (UCP2), a protein involved in the control of mitochondrial membrane potential, and four-and-a-half LIM domain protein-1 (FHL1), a member of the LIM protein family, were predicted to be upregulated. Upregulation of these genes was confirmed by quantitative reverse transcriptase-polymerase chain reaction at all time points tested during the development of cardiomyopathy in mice overexpressing Gsalpha. In mice overexpressing beta1AR, beta2AR, or protein kinase A, increased UCP2 and FHL1 expression was also observed at the onset of cardiomyopathy. BetaAR blockade treatment reversed the cardiomyopathy and suppressed the increased expression of UCP2 and FHL1 in mice overexpressing Gsalpha. CONCLUSIONS: UCP2 and FHL1 are important candidate genes that correlate with the development of betaAR-induced cardiomyopathy in different mouse models with enhanced betaAR signaling. In addition to preserving cardiac function, betaAR blockade treatment also prevents the genomic regulation that correlates with the onset of heart failure.

Flow loading induces macrophage antioxidative gene expression in experimental aneurysms.

OBJECTIVE: Reactive oxygen species may act as proinflammatory mediators in abdominal aortic aneurysm (AAA) disease. Flow loading increases antioxidative enzyme expression and limits reactive oxygen species production in vascular smooth muscle cells in vitro, limits experimental AAA enlargement in rodent models, and is indirectly associated with reduced clinical AAA risk. We attempted to determine the mechanism or mechanisms by which flow loading limits AAA enlargement. METHODS AND RESULTS: Rodent AAAs were flow loaded via femoral arteriovenous fistula creation. Aortic wall shear stress and relative wall strain were significantly higher in flow-loaded rodents. Flow loading reduced AAA diameter by 26% despite evidence of flow-mediated aortic enlargement proximal to the aneurysmal segment. Messenger RNA from AAA tissue was harvested for cDNA labeling and hybridization to a 384-clone DNA microarray. Twenty-nine genes were differentially expressed (relative intensity/relative intensity of control ratio >1.5 and <0.67) in flow-loaded compared with normal flow AAA tissue, including heme oxygenase 1 (HO-1). Increased HO-1 expression was confirmed via reverse transcriptase-polymerase chain reaction. Immunohistochemistry localized HO-1 expression to infiltrative macrophages. alpha-Tocopherol was found to be as effective as flow loading in limiting AAA enlargement. Flow loading and alpha-tocopherol therapy reduced AAA reactive oxygen species production. CONCLUSIONS: Flow loading may attenuate AAA enlargement via wall shear or strain-related reductions in oxidative stress.

Characterization of pDJA1, a cardiac-specific chaperone found by genomic profiling of the post-ischemic swine heart.

BACKGROUND: Previously, we showed by subtractive hybridization in a swine model of ischemia/reperfusion that an upregulation of genes participating in mechanisms of cell survival is a potential genomic mechanism to tilt the balance from necrosis to functional reversibility. METHODS AND RESULTS: We present here the full-length sequencing and characterization of a novel gene that was found in this subtraction, encoding a cardiac-specific DnaJ-like co-chaperone that we call Pig DnaJ-like protein A1 (pDJA1). The expression of pDJA1 was found to be restricted to the heart, as opposed to skeletal muscle, liver, lung, kidney, aorta, stomach and spleen. Expression of pDJA1 is restricted to cardiac myocytes, as determined by in situ hybridization. The transcript is expressed more in the left ventricle than in the other cardiac chambers. Remarkably, expression of pDJA1 follows a transmural gradient in the left ventricle, with the highest level of expression in the subendocardium. Expression of pDJA1 slightly increased during an episode of ischemia, but increased by 4-fold during the following period of reperfusion. Adenovirus-mediated transduction of pDJA1 in isolated rat neonatal cardiac myocytes decreased by 65% the rate of apoptosis induced by staurosporine. CONCLUSION: Therefore, pDJA1 is a novel heart-specific, ventricle-enriched cardioprotective co-chaperone, which participates in the program of cell survival that limits irreversible damage in post-ischemic myocardium.

Inducible and selective transgene expression in murine vascular endothelium.

We have developed a system utilizing the murine Tie2 promoter/enhancer coupled with the "tetracycline-on" regulatory elements to create a model that allows regulated and selective expression of a beta-galactosidase (betaGal) reporter transgene in the adult murine vascular endothelium. Two independent lines of viable and fertile mice were characterized, and they exhibit minimal betaGal expression under basal conditions. In response to exogenous doxycycline (Dox), selective expression of betaGal was demonstrated in the vascular endothelium of all tissues examined. En face analyses of the aorta and its principle branches indicate that the vast majority of lumenal endothelial cells express the transgene. Inducible betaGal expression also extends to the endocardium and the microvasculature of all organs. There is no evidence of specific transgene expression in nonendothelial cell types. Induction of the betaGal was effectively achieved after 3 days of oral Dox treatment and persisted for over 3 mo with continuous administration. This model can now be widely applied to study the role of specific genes in the phenotype of adult murine vasculature.

Gene expression profile of human endothelial cells exposed to sustained fluid shear stress.

Biomechanical forces can modulate endothelial phenotype through changes in gene expression. We hypothesized that physiological laminar shear stresses (LSS) act as differentiative stimuli on endothelial cells (EC) to alter gene expression, creating an antioxidant, anti-apoptotic and anti-proliferative environment. The transcriptional profile of cultured human umbilical vein endothelial cells (HUVEC) exposed to LSS was evaluated by GeneCalling; 107 genes demonstrated at least a twofold change in expression at 24 h (LSS vs. static). These flow-responsive genes represent a limited number of functional clusters that include transcription factors, antioxidants, signaling molecules, cell cycle regulators, and genes involved in cellular differentiation. Immunohistochemistry and in situ hybridization confirmed that many of these flow-responsive genes, including the novel basic helix-loop-helix transcription factor Hath6, are expressed in EC in vivo. Thus these data identify a limited set of flow-responsive genes expressed in the endothelium that may be responsible for the establishment and maintenance of the flow-adapted endothelial phenotype in vivo.

Genetic endothelial systems biology of sickle stroke risk.

Genetic differences in endothelial biology could underlie development of phenotypic heterogeneity among persons afflicted with vascular diseases. We obtained blood outgrowth endothelial cells from 20 subjects with sickle cell anemia (age, 4-19 years) shown to be either at-risk (n=11) or not-at-risk (n=9) for ischemic stroke because of, respectively, having or not having occlusive disease at the circle of Willis. Gene expression profiling identified no significant single gene differences between the 2 groups, as expected. However, analysis of Biological Systems Scores, using gene sets that were predetermined to survey each of 9 biologic systems, showed that only changes in inflammation signaling are characteristic of the at-risk subjects, as supported by multiple statistical approaches. Correspondingly, subsequent biologic testing showed significantly exaggerated RelA activation on the part of blood outgrowth endothelial cells from the at-risk subjects in response to stimulation with interleukin-1beta/tumor necrosis factoralpha. We conclude that the pathobiology of circle of Willis disease in the child with sickle cell anemia predominantly involves inflammation biology, which could reflect differences in genetically determined endothelial biology that account for differing host responses to inflammation.

New insights into endothelial diversity.

It has long been appreciated that endothelium from distinct vascular beds manifest diverse structural and functional properties. With current technologies, we have begun to systematically define the unique molecular profiles that orchestrate and sustain this diversity, and to assess the influences of various environmental and developmental stimuli on these processes. This review focuses on recent advances and findings that provide insights into both the extent and causes of endothelial cell diversity.

Adaptation of the endothelium to fluid flow: in vitro analyses of gene expression and in vivo implications.

Biomechanical forces generated by blood flow play an important role in the pathogenesis of vascular disease. For example, regions exposed to non-uniform shear stresses develop early atherosclerotic lesions while areas exposed to uniform shear stresses are protected. A variety of in vitro flow apparatuses have been created to apply well-characterized flow patterns to endothelial cells in an effort to dissect the cellular and molecular pathways involved in these distinct processes. Recent advances in biotechnology have permitted large-scale transcriptional profiling techniques to replace candidate gene screens and have allowed the genome-wide examination of biomechanical force-induced endothelial gene expression profiles. This review provides an overview of biomechanical force-induced modulation of endothelial phenotype. It examines the effect of sustained laminar shear stress (LSS), a type of uniform shear stress, on in vitro endothelial gene expression by synthesizing data from the early candidate gene and differential display polymerase chain reaction (PCR) approaches to the numerous, recent, high throughput functional genomic analyses. These studies demonstrate that prolonged LSS regulates the expression of only a small percentage (approximately 1-5%) of endothelial genes, and this transcriptional profile produces an endothelial phenotype that is quiescent, being protected from apoptosis, inflammation and oxidative stress. These observations provide a possible molecular mechanism for the strong correlation between patterns of blood flow and the occurrence of vascular pathologies, such as atherosclerosis, in vivo.

A novel secreted, cell-surface glycoprotein containing multiple epidermal growth factor-like repeats and one CUB domain is highly expressed in primary osteoblasts and bones.

We have previously identified a novel family of secreted, cell-surface proteins expressed in human vascular endothelium. To date, two family members have been described, sharing a characteristic domain structure including an amino-terminal signal peptide followed by multiple copies of epidermal growth factor (EGF)-like repeats, a spacer region, and one CUB domain at the carboxyl terminus. Thus, this family was termed SCUBE for signal peptide-CUB-EGF-like domain containing proteins. Here we described the identification and characterization of one additional member of the SCUBE family named SCUBE3 in humans, sharing an overall 60% protein identity and a similar domain structure with other family members. Real-time quantitative reverse transcriptase-PCR and Northern blot analyses revealed that this gene is highly expressed in primary osteoblasts and the long bones (humerus and femur), followed by a low level of expression in human umbilical vein endothelial cells and in heart. When overexpressed in human embryonic kidney 293T cells, the recombinant hSCUBE3 protein is a secreted glycoprotein that can form oligomers tethered to the cell surface. Interestingly, the secreted hSCUBE3 protein can be further proteolytically processed by a serum-associated protease to release the EGF-like repeats from the CUB domain. The SCUBE3 gene is mapped to human chromosome 6p21.3, a region that has been linked with the locus for a rare form of metabolic bone disease, Paget's disease of bone 1. Together, this novel secreted, cell-surface osteoblast protein may act locally and/or distantly through a proteolytic mechanism, and may play an important role in bone cell biology.

A novel interleukin-17 receptor-like protein identified in human umbilical vein endothelial cells antagonizes basic fibroblast growth factor-induced signaling.

We have previously utilized a combination of high throughput sequencing and genome-wide microarray profiling analyses to identify novel cell-surface proteins expressed in human umbilical vein endothelial cells. One gene identified by this approach encodes a type I transmembrane receptor that shares sequence homology with the intracellular domain of members of the interleukin-17 (IL-17) receptor family. Real-time quantitative PCR and Northern analyses revealed that this gene is highly expressed in human umbilical vein endothelial cells and in several highly vascularized tissues such as kidney, colon, skeletal muscle, heart, and small intestine. In addition, we also found that it is also highly expressed in the ductal epithelial cells of human salivary glands, seminal vesicles, and the collecting tubules of the kidney by in situ hybridization. This putative receptor, which we have termed human SEF (hSEF), is also expressed in a variety of breast cancer tissues. In co-immunoprecipitation assays, this receptor is capable of forming homomeric complexes and can interact with fibroblast growth factor (FGF) receptor 1. Overexpression of this receptor inhibits FGF induction of an FGF-responsive reporter gene in human 293T cells. This appears to occur as a result of specific inhibition of p42/p44 ERK in the absence of upstream MEK inhibition. This inhibitory effect is dependent upon a functional intracellular domain since deletion mutants missing the IL-17 receptor-like domain lack this inhibitory effect. These findings are consistent with the recent discovery of the zebrafish homologue, Sef (similar expression to fgf genes), which specifically antagonizes FGF signaling when ectopically expressed in zebrafish or Xenopus laevis embryos. Based on sequence and functional similarities, this novel IL-17 receptor homologue represents a potential human SEF and is likely to play critical roles in endothelial or epithelial functions such as proliferation, migration, and angiogenesis.

Identification of a novel family of cell-surface proteins expressed in human vascular endothelium.

Vascular endothelial cells (EC) play a key role in a variety of pathophysiologic processes, such as angiogenesis, inflammation, cancer metastasis, and vascular diseases. As part of a strategy to identify all genes expressed in human EC, a full-length cDNA encoding a potential secreted protein harboring 10 epidermal growth factor (EGF)-like domains and one CUB domain at the carboxyl terminus (termed, SCUBE1 for Signal peptide-CUB-EGF-like domain containing protein 1) was identified. SCUBE1 shares homology with several protein families, including members of the fibrillin and Notch families, and the anticoagulant proteins, thrombomodulin and protein C. SCUBE1 mRNA is found in several highly vascularized tissues such as liver, kidney, lung, spleen, and brain and is selectively expressed in EC by in situ hybridization. SCUBE1 is a secreted glycoprotein that can form oligomers and manifests a stable association with the cell surface. A second gene encoding a homologue (designated SCUBE2) was also identified and is expressed in EC as well as other cell types. SCUBE2 is also a cell-surface protein and can form a heteromeric complex with SCUBE1. Both SCUBE1 and SCUBE2 are rapidly down-regulated in EC after interleukin-1beta and tumor necrosis factor-alpha treatment in vitro and after lipopolysaccharide injection in vivo. Thus, SCUBE1 and SCUBE2 define an emerging family of human secreted proteins that are expressed in vascular endothelium and may play important roles in development, inflammation, and thrombosis.