Michael R. O'Dell

Cyclophilin A enhances vascular oxidative stress and the development of angiotensin II–induced aortic aneurysms

Inflammation and oxidative stress are pathogenic mediators of many diseases, but molecules that could be therapeutic targets remain elusive. Inflammation and matrix degradation in the vasculature are crucial for abdominal aortic aneurysm (AAA) formation. Cyclophilin A (CypA, encoded by Ppia) is highly expressed in vascular smooth muscle cells (VSMCs), is secreted in response to reactive oxygen species (ROS) and promotes inflammation. Using the angiotensin II (AngII)-induced AAA model in Apoe−/− mice, we show that Apoe−/−Ppia−/− mice are completely protected from AngII–induced AAA formation, in contrast to Apoe−/−Ppia+/+ mice. Apoe−/−Ppia−/− mice show decreased inflammatory cytokine expression, elastic lamina degradation and aortic expansion. These features were not altered by reconstitution of bone marrow cells from Ppia+/+ mice. Mechanistic studies showed that VSMC-derived intracellular and extracellular CypA are required for ROS generation and matrix metalloproteinase-2 activation. These data define a previously undescribed role for CypA in AAA formation and suggest CypA as a new target for treating cardiovascular disease.

Cyclophilin A Mediates Vascular Remodeling by Promoting Inflammation and Vascular Smooth Muscle Cell Proliferation

Background— Oxidative stress, generated by excessive reactive oxygen species, promotes cardiovascular disease. Cyclophilin A (CyPA) is a 20-kDa chaperone protein secreted from vascular smooth muscle cells (VSMCs) in response to reactive oxygen species that stimulates VSMC proliferation and inflammatory cell migration in vitro; however, the role CyPA plays in vascular function in vivo remains unknown. Methods and Results— We tested the hypothesis that CyPA contributes to vascular remodeling by analyzing the response to complete carotid ligation in CyPA knockout mice, wild-type mice, and mice that overexpress CyPA in VSMC (VSMC-Tg). After carotid ligation, CyPA expression in vessels of wild-type mice increased dramatically and was significantly greater in VSMC-Tg mice. Reactive oxygen species–induced secretion of CyPA from mouse VSMCs correlated significantly with intracellular CyPA expression. Intimal and medial hyperplasia correlated significantly with CyPA expression after 2 weeks of carotid ligation, with marked decreases in CyPA knockout mice and increases in VSMC-Tg mice. Inflammatory cell migration into the intima was significantly reduced in CyPA knockout mice and increased in VSMC-Tg mice. Additionally, VSMC proliferation assessed by Ki67+ cells was significantly less in CyPA knockout mice and was increased in VSMC-Tg mice. The importance of CyPA for intimal and medial thickening was shown by strong correlations between CyPA expression and the number of both inflammatory cells and proliferating VSMCs in vivo and in vitro. Conclusions— In response to low flow, CyPA plays a crucial role in VSMC migration and proliferation, as well as inflammatory cell accumulation, thereby regulating flow-mediated vascular remodeling and intima formation.

Cyclophilin A is an inflammatory mediator that promotes atherosclerosis in apolipoprotein E–deficient mice

Cyclophilin A promotes atherosclerosis in part by inducing reactive oxygen species and promoting endothelial cell apoptosis and macrophage recruitment into lesions.

Kras(G12D) and p53 mutation cause primary intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (IHCC) is a primary cancer of the liver with an increasing incidence and poor prognosis. Preclinical studies of the etiology and treatment of this disease are hampered by the relatively small number of available IHCC cell lines or genetically faithful animal models. Here we report the development of a genetically engineered mouse model of IHCC that incorporates two of the most common mutations in human IHCC, activating mutations of Kras (Kras(G12D)) and deletion of p53. Tissue-specific activation of Kras(G12D) alone resulted in the development of invasive IHCC with low penetrance and long latency. Latency was shortened by combining Kras(G12D) activation with heterozygous or homozygous deletion of p53 (mean survival of 56 weeks vs. 19 weeks, respectively), which also resulted in widespread local and distant metastasis. Serial analysis showed that the murine models closely recapitulated the multistage histopathologic progression of the human disease, including the development of stroma-rich tumors and the premalignant biliary lesions, intraductal papillary biliary neoplasms (IPBN), and Von Meyenburg complexes (VMC; also known as biliary hamartomas). These findings establish a new genetically and histopathologically faithful model of IHCC and lend experimental support to the hypothesis that IPBN and VMC are precursors to invasive cancers.

PKCζ decreases eNOS protein stability via inhibitory phosphorylation of ERK5

PKCζ has emerged as a pathologic mediator of endothelial cell dysfunction, based on its essential role in tumor necrosis factor α (TNFα)-mediated inflammation. In contrast, extracellular signal-regulated kinase 5 (ERK5) function is required for endothelial cell homeostasis as shown by activation of Krüppel-like factor 2 (KLF2), increased endothelial nitric-oxide synthase (eNOS) expression, and inhibition of apoptosis. We hypothesized that protein kinase C ζ (PKCζ) activation by TNFα would inhibit the ERK5/KLF2/eNOS pathway. TNFα inhibited the steady laminar flow-induced eNOS expression, and this effect was reversed by the dominant-negative form of PKCζ (Ad.DN-PKCζ). In addition, ERK5 function was inhibited by either TNFα or the transfection of the catalytic domain of PKCζ. This inhibition was reversed by PKCζ small interfering RNA. PKCζ was found to bind to ERK5 under basal conditions with coimmunoprecipitation and the mammalian 2-hybrid assay. Furthermore, PKCζ phosphorylates ERK5, and mutation analysis showed that the preferred site is S486. Most importantly, we found that the predominant effect of TNFα stimulation of PKCζ was to decrease eNOS protein stability that was recapitulated by transfecting Ad.ERK5S486A mutant. Finally, aortic en face analysis of ERK5/PKCζ activity showed high PKCζ and ERK5 staining in the athero-prone region. Taken together our results show that PKCζ binds and phosphorylates ERK5, thereby decreasing eNOS protein stability and contributing to early events of atherosclerosis.

Cyclophilin A Promotes Cardiac Hypertrophy in Apolipoprotein E–Deficient Mice

Objective—Cyclophilin A (CyPA, encoded by Ppia) is a proinflammatory protein secreted in response to oxidative stress in mice and humans. We recently demonstrated that CyPA increased angiotensin II (Ang II)–induced reactive oxygen species (ROS) production in the aortas of apolipoprotein E (Apoe)−/− mice. In this study, we sought to evaluate the role of CyPA in Ang II–induced cardiac hypertrophy. Methods and Results—Cardiac hypertrophy was not significantly different between Ppia+/+ and Ppia−/− mice infused with Ang II (1000 ng/min per kg for 4 weeks). Therefore, we investigated the effect of CyPA under conditions of high ROS and inflammation using the Apoe−/− mice. In contrast to Apoe−/− mice, Apoe−/−Ppia−/− mice exhibited significantly less Ang II–induced cardiac hypertrophy. Bone marrow cell transplantation showed that CyPA in cells intrinsic to the heart plays an important role in the cardiac hypertrophic response. Ang II–induced ROS production, cardiac fibroblast proliferation, and cardiac fibroblast migration were markedly decreased in Apoe−/−Ppia−/− cardiac fibroblasts. Furthermore, CyPA directly induced the hypertrophy of cultured neonatal cardiac myocytes. Conclusion—CyPA is required for Ang II–mediated cardiac hypertrophy by directly potentiating ROS production, stimulating the proliferation and migration of cardiac fibroblasts, and promoting cardiac myocyte hypertrophy.

TGF-β and αvβ6 Integrin Act in a Common Pathway to Suppress Pancreatic Cancer Progression

The TGF-β pathway is under active consideration as a cancer drug target based on its capacity to promote cancer cell invasion and to create a protumorigenic microenvironment. However, the clinical application of TGF-β inhibitors remains uncertain as genetic studies show a tumor suppressor function of TGF-β in pancreatic cancer and other epithelial malignancies. Here, we used genetically engineered mouse models to investigate the therapeutic impact of global TGF-β inhibition in pancreatic cancer in relation to tumor stage, genetic profile, and concurrent chemotherapy. We found that αvβ6 integrin acted as a key upstream activator of TGF-β in evolving pancreatic cancers. In addition, TGF-β or αvβ6 blockade increased tumor cell proliferation and accelerated both early and later disease stages. These effects were dependent on the presence of Smad4, a central mediator of TGF-β signaling. Therefore, our findings indicate that αvβ6 and TGF-β act in a common tumor suppressor pathway whose pharmacologic inactivation promotes pancreatic cancer progression.

Impaired spine formation and learning in GPCR kinase 2 interacting protein-1 (GIT1) knockout mice

The G-protein coupled receptor (GPCR)-kinase interacting proteins 1 and 2 (GIT1 and GIT2) are scaffold proteins with ADP-ribosylating factor GTPase activity. GIT1 and GIT2 control numerous cellular functions and are highly expressed in neurons, endothelial cells and vascular smooth muscle cells. GIT1 promotes dendritic spine formation, growth and motility in cultured neurons, but its role in brain in vivo is unknown. By using global GIT1 knockout mice (GIT1 KO), we show that compared to WT controls, deletion of GIT1 results in markedly reduced dendritic length and spine density in the hippocampus by 36.7% (p<0.0106) and 35.1% (p<0.0028), respectively. This correlated with their poor adaptation to new environments as shown by impaired performance on tasks dependent on learning. We also studied the effect of GIT1 gene deletion on brain microcirculation. In contrast to findings in systemic circulation, GIT1 KO mice had an intact blood-brain barrier and normal regional cerebral blood flow as determined with radiotracers. Thus, our data suggest that GIT1 plays an important role in brain in vivo by regulating spine density involved in synaptic plasticity that is required for processes involved in learning.

G-Protein–Coupled Receptor Kinase Interacting Protein-1 Is Required for Pulmonary Vascular Development

Background— The G-protein–coupled receptor kinase interacting protein-1 (GIT1) is a multidomain scaffold protein that participates in many cellular functions including receptor internalization, focal adhesion remodeling, and signaling by both G-protein–coupled receptors and tyrosine kinase receptors. However, there have been no in vivo studies of GIT1 function to date. Methods and Results— To determine essential functions of GIT1 in vivo, we generated a traditional GIT1 knockout mouse. GIT1 knockout mice exhibited ≈60% perinatal mortality. Pathological examination showed that the major abnormality in GIT1 knockout mice was impaired lung development characterized by markedly reduced numbers of pulmonary blood vessels and increased alveolar spaces. Given that vascular endothelial growth factor (VEGF) is essential for pulmonary vascular development, we investigated the role of GIT1 in VEGF signaling in the lung and cultured endothelial cells. Because activation of phospholipase-Cγ (PLCγ) and extracellular signal-regulated kinases 1/2 (ERK1/2) by angiotensin II requires GIT1, we hypothesized that GIT1 mediates VEGF-dependent pulmonary angiogenesis by modulating PLCγ and ERK1/2 activity in endothelial cells. In cultured endothelial cells, knockdown of GIT1 decreased VEGF-mediated phosphorylation of PLCγ and ERK1/2. PLCγ and ERK1/2 activity in lungs from GIT1 knockout mice was reduced postnatally. Conclusions— Our data support a critical role for GIT1 in pulmonary vascular development by regulating VEGF-induced PLCγ and ERK1/2 activation.

Abstract PR09: Plac8 links oncogenic mutations to regulation of autophagy and is critical to pancreatic cancer progression

A) Pancreatic ductal adenocarcinoma (PDA) depends on a marked reprogramming of metabolic pathways, including the acquisition of autophagy dependence, for survival and growth. How common mutations in PDA cause autophagy dependence as well as the timing of autophagy activation in the course of cancer progression, have not been established. Here we show how Plac8, a gene synergistically up-regulated in response to the common cooperating oncogenic mutations found in PDA (RAS activation and functional loss of p53), is critical to the growth of PDA by sustaining autophagy via facititating autophagosome-lysosome fusion. Furthermore, we delineate that Kras and p53 mutations cooperate to induce autophagic flux. B) To establish Plac8’s role in PDA growth and autophagy we use murine cell lines and human PDA cell lines to determine 1) the lysosomal localization of Plac8, 2) its role in regulating autophagy using both loss of function and gain of function approaches, 3) the impact on lysosomal biology, and 4) the relationship between Plac8 and other genetic pathways governing autophagy. Using genetically engineered models of PDA we determine the timing of autophagy activation in PDA progression and the impact of Plac8 mutation. C) We identify Plac8 as a novel regulator of autophagosome-lysosome fusion required for PDA growth, thus providing a mechanistic link between oncogenic mutations and the activation of autophagy in cancer. Plac8 expression is required for growth of human PDA cells as xenografts in mice, as well as activation of autophagy. We find that concurrent mutation of KRAS and p53 is critical for maximal induction of autophagy in vitro. Correspondingly, using genetically engineered mouse models of PDA (Pdx1-Cre; LSL-KrasG12D; p53L/+), in which loss of p53 function occurs in a step-wise manner relying on the spontaneous loss of a heterozygous WT p53 allele, we see a step-wise incremental increase in LC3 puncta in vivo with each histological stage through the course of PDA progression. Thus, we find that the cooperative effects of KRAS and p53 drive activation of autophagy rather than either mutation alone. The overall survival of a Pdx1-Cre; LSL-KrasG12D; p53L/+; Plac8null murine cohort (OS 27.9 wks) was significantly longer than a Pdx1-Cre; LSL-KrasG12D; p53L/+; Plac8wt cohort (OS 17.0 wks, p=0.0006) demonstrating in vivo that genetic inactivation of Plac8 impedes cancer progression and resulting death. Our data suggest that the role of Plac8 in facilitating autophagy is critical to cancer, as the requirement of Plac8 for both tumorigenicity and autophagy can be compensated by over-expression of Atg12, a gene critical for autophagosome formation or by constitutively activated Rab7, a gene encoding a GTP-binding protein stimulating autophagosome-lysosome fusion. D) We conclude that Plac8 may offer a potential therapeutic window and point of intervention, as Plac8 mutation in the engineered PDA model inhibits cancer progression and significantly improves survival while having a minimal impact on the overall fitness of the animals. In fact, Plac8, and regulation of autophagosome-lysosome fusion, has specific relevance to regulation of autophagy during malignant cell transformation as Plac8 and the processes it regulates, appear to be largely dispensable to many normal physiologic processes. This abstract is also presented as Poster B36. Citation Format: Vijaya Balakrishnan, Kinsey Conan, Michael O9Dell, Jing Li Huang, Laurel Newman, Christa Whitney-Miller, Hartmut Land, Aram Hezel. Plac8 links oncogenic mutations to regulation of autophagy and is critical to pancreatic cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr PR09.