Publications

The dynamic interactions between tumor endothelial cells (TECs) and the immune microenvironment play a critical role in the progression of non-small cell lung cancer (NSCLC). In general, endothelial cells exhibit diverse immunomodulatory properties, influencing immune cell recruitment, antigen presentation, and regulation of immune checkpoint expression. Understanding the multifaceted roles of TECs as well as assigning specific functional hallmarks to various TEC phenotypes offer new avenues for targeted development of therapeutic interventions, particularly in the context of advanced immunotherapy and anti-angiogenic treatments. This review provides insights into the complex interplay between TECs and the immune system in NSCLC including discussion of potential optimized therapeutic opportunities.

In clinical trials, aldosterone antagonists reduce cardiovascular ischemia and mortality by unknown mechanisms. Aldosterone is a steroid hormone that signals through renal mineralocorticoid receptors (MRs) to regulate blood pressure. MRs are expressed and regulate gene transcription in human vascular cells, suggesting that aldosterone might have direct vascular effects. Using gene expression profiling, we identify the pro-proliferative VEGF family member placental growth factor (PGF) as an aldosterone-regulated vascular MR target gene in mice and humans. Aldosterone-activated vascular MR stimulated Pgf gene transcription and increased PGF protein expression and secretion in the mouse vasculature. In mouse vessels with endothelial damage and human vessels from patients with atherosclerosis, aldosterone enhanced expression of PGF and its receptor, FMS-like tyrosine kinase 1 (Flt1). In atherosclerotic human vessels, MR antagonists inhibited PGF expression. In vivo, aldosterone infusion augmented vascular remodeling in mouse carotids following wire injury, an effect that was lost in Pgf-/- mice. In summary, we have identified PGF as what we believe to be a novel downstream target of vascular MR that mediates aldosterone augmentation of vascular injury. These findings suggest a non-renal mechanism for the vascular protective effects of aldosterone antagonists in humans and support targeting the vascular aldosterone/MR/PGF/Flt1 pathway as a therapeutic strategy for ischemic cardiovascular disease.

Background Aldosterone levels correlate with the incidence of myocardial infarction and mortality in cardiovascular patients. Aldosterone promotes atherosclerosis in animal models, but the mechanisms are poorly understood. Methods and Results Aldosterone was infused to achieve pathologically relevant levels that did not increase blood pressure in the atherosclerosis‐prone apolipoprotein E–knockout mouse (ApoE−/−). Aldosterone increased atherosclerosis in the aortic root 1.8±0.1‐fold after 4 weeks and in the aortic arch 3.7±0.2‐fold after 8 weeks, without significantly affecting plaque size in the abdominal aorta or traditional cardiac risk factors. Aldosterone treatment increased lipid content of plaques (2.1±0.2‐fold) and inflammatory cell content (2.2±0.3‐fold), induced early T‐cell (2.9±0.3‐fold) and monocyte (2.3±0.3‐fold) infiltration into atherosclerosis‐prone vascular regions, and enhanced systemic inflammation with increased spleen weight (1.52±0.06‐fold) and the circulating cytokine RANTES (regulated and normal T cell secreted; 1.6±0.1‐fold). To explore the mechanism, 7 genes were examined for aldosterone regulation in the ApoE−/− aorta. Further studies focused on the proinflammatory placental growth factor (Pl GF ), which was released from aldosterone‐treated ApoE−/− vessels. Activation of the mineralocorticoid receptor by aldosterone in human coronary artery smooth muscle cells ( SMC s) caused the release of factors that promote monocyte chemotaxis, which was inhibited by blocking monocyte Pl GF receptors. Furthermore, Pl GF ‐deficient ApoE−/− mice were resistant to early aldosterone‐induced increases in plaque burden and inflammation. Conclusions Aldosterone increases early atherosclerosis in regions of turbulent blood flow and promotes an inflammatory plaque phenotype that is associated with rupture in humans. The mechanism may involve SMC release of soluble factors that recruit activated leukocytes to the vessel wall via Pl GF signaling. These findings identify a novel mechanism and potential treatment target for aldosterone‐induced ischemia in humans.

Rationale: Paracrine growth factor-mediated crosstalk between cardiac myocytes and nonmyocytes in the heart is critical for programming adaptive cardiac hypertrophy in which myocyte size, capillary density, and the extracellular matrix function coordinately. Objective: To examine the role that placental growth factor (PGF) plays in the heart as a paracrine regulator of cardiac adaptation to stress stimulation. Methods and Results: PGF is induced in the heart after pressure-overload stimulation, where it is expressed in both myocytes and nonmyocytes. We generated cardiac-specific and adult inducible PGF-overexpressing transgenic mice and analyzed Pgf −/− mice to examine the role that this factor plays in cardiac disease and paracrine signaling. Although PGF transgenic mice did not have a baseline phenotype or a change in capillary density, they did exhibit a greater cardiac hypertrophic response, a greater increase in capillary density, and increased fibroblast content in the heart in response to pressure-overload stimulation. PGF transgenic mice showed a more adaptive type of cardiac growth that was protective against signs of failure with pressure overload and neuroendocrine stimulation. Antithetically, Pgf −/− mice rapidly died of heart failure within 1 week of pressure overload, they showed an inability to upregulate angiogenesis, and they showed significantly less fibroblast activity in the heart. Mechanistically, we show that PGF does not have a direct effect on cardiomyocytes but works through endothelial cells and fibroblasts by inducing capillary growth and fibroblast proliferation, which secondarily support greater cardiac hypertrophy through intermediate paracrine growth factors such as interleukin-6. Conclusions: PGF is a secreted factor that supports hypertrophy and cardiac function during pressure overload by affecting endothelial cells and fibroblasts that in turn stimulate and support the myocytes through additional paracrine factors.

Growth arrest–specific protein 6 (Gas 6) is involved in inflammatory kidney diseases, vascular remodeling, cell adhesion, and thrombus formation. We explored a role for Gas 6 in aldosterone-induced target organ damage. We observed that Gas 6 was upregulated in rats with high aldosterone levels. Mineralocorticoid receptor blockade prevented target organ damage and decreased the elevated Gas 6 expression. Vascular smooth muscle cells given aldosterone increased their Gas 6 expression in vitro. To test the pathophysiological relevance, we investigated the effects of deoxycorticosterone acetate (DOCA) on Gas 6 gene-deleted ( −/− ) mice. After 6 weeks DOCA, Gas 6 −/− mice developed similar telemetric blood pressure elevations compared to wild-type mice but were protected from cardiac hypertrophy. Cardiac expression of interleukin 6 and collagen IV was blunted in Gas 6 −/− mice, indicating reduced inflammation and fibrosis. Gas 6 −/− mice also had an improved renal function with reduced albuminuria, compared to wild-type mice. Renal fibrosis and fibronectin deposition in the kidney were also reduced. Gas 6 deficiency reduces the detrimental effects of aldosterone on cardiac and renal remodeling independent of blood pressure reduction. Gas 6 appears to play a role in mineralocorticoid receptor-mediated target organ damage. Furthermore, because warfarin interferes with Gas 6 protein expression, the findings could be of clinical relevance for anticoagulant choices.

Reactive astrocytes are thought to protect the penumbra during brain ischemia, but direct evidence has been lacking due to the absence of suitable experimental models. Previously, we generated mice deficient in two intermediate filament (IF) proteins, glial fibrillary acidic protein (GFAP) and vimentin, whose upregulation is the hallmark of reactive astrocytes. GFAP −/− Vim −/− mice exhibit attenuated posttraumatic reactive gliosis, improved integration of neural grafts, and posttraumatic regeneration. Seven days after middle cerebral artery (MCA) transection, infarct volume was 210 to 350% higher in GFAP −/− Vim −/− than in wild-type (WT) mice; GFAP −/− , Vim −/− and WT mice had the same infarct volume. Endothelin B receptor (ET B R) immunoreactivity was strong on cultured astrocytes and reactive astrocytes around infarct in WT mice but undetectable in GFAP −/− Vim −/− astrocytes. In WT astrocytes, ET B R colocalized extensively with bundles of IFs. GFAP −/− Vim −/− astrocytes showed attenuated endothelin-3-induced blockage of gap junctions. Total and glutamate transporter-1 (GLT-1)-mediated glutamate transport was lower in GFAP −/− Vim −/− than in WT mice. DNA array analysis and quantitative real-time PCR showed downregulation of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of tissue plasminogen activator. Thus, reactive astrocytes have a protective role in brain ischemia, and the absence of astrocyte IFs is linked to changes in glutamate transport, ET B R-mediated control of gap junctions, and PAI-1 expression.

Purpose: Macrophage specific knockout of PHD-2 prolylhydroxylase domain protein 2 (PHD-2-/-), the crucial cellular oxygen sensor, increased anti-inflammatory M2 macrophages, angiogenesis and arteriogenesis in mice. As macrophages and angiogenesis are known to enhance progression of atherosclerotic plaques, we hypothesized that macrophage PHD-2-/- would increase plaque size and progression.

Plasminogen activators urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) are extracellular proteases involved in various tissue remodeling processes. A requirement for uPA activity in skeletal myogenesis was recently demonstrated in vitro. The role of plasminogen activators in skeletal muscle regeneration in vivo in wild-type, uPA-deficient, and tPA-deficient mice is investigated here. Wild-type and tPA−/− mice completely repaired experimentally damaged skeletal muscle. In contrast, uPA−/− mice had a severe regeneration defect, with decreased recruitment of blood-derived monocytes to the site of injury and with persistent myotube degeneration. In addition, uPA-deficient mice accumulated fibrin in the degenerating muscle fibers; however, the defibrinogenation of uPA-deficient mice resulted in a correction of the muscle regeneration defect. A similar severe regeneration deficit with persistent fibrin deposition was also reproducible in plasminogen-deficient mice after injury, suggesting that fibrinolysis by uPA-mediated plasminogen activation plays a fundamental role in skeletal muscle regeneration. In conclusion, the uPA-plasmin system is identified as a critical component of the mammalian skeletal muscle regeneration process, possibly because it prevents intramuscular fibrin accumulation and contributes to the adequate inflammatory response after injury. These studies demonstrate the requirement of an extracellular proteolytic cascade during muscle regeneration in vivo.

Abstract Protein phosphatase 2A (PP2A) complexes counteract many oncogenic kinase pathways. In cancer cells, PP2A function can be compromised by several mechanisms, including sporadic mutations in its scaffolding A and regulatory B subunits or more frequently through overexpression of cellular PP2A inhibitors. Here, we identify a novel genetic mechanism by which PP2A function is recurrently affected in human cancer, involving haploinsufficiency of PPP2R4, a gene encoding the cellular PP2A activator PTPA. Notably, up to 70% of cancer patients showed a heterozygous deletion or missense mutations in PPP2R4. Cancer-associated PTPA mutants exhibited decreased abilities to bind the PP2A-C subunit or activate PP2A and failed to reverse the tumorigenic phenotype induced by PTPA suppression, indicating they function as null alleles. In Ppp2r4 gene-trapped (gt) mice showing residual PTPA expression, total PP2A activity and methylation were reduced, selectively affecting specific PP2A holoenzymes. Both PTPAgt/gt and PTPA+/gt mice showed higher rates of spontaneous tumors, mainly hematologic malignancies and hepatocellular adenomas and carcinomas. These tumors exhibited increased c-Myc phosphorylation and increased Wnt or Hedgehog signaling. We observed a significant reduction in lifespan in PTPA+/gt mice compared with wild-type mice. In addition, chemical-induced skin carcinogenesis was accelerated in PTPA+/gt compared with wild-type mice. Our results provide evidence for PPP2R4 as a haploinsufficient tumor suppressor gene, defining a high-penetrance genetic mechanism for PP2A inhibition in human cancer. Cancer Res; 77(24); 6825–37. ©2017 AACR.

<b>Rationale:</b> Chronic obstructive pulmonary disease (COPD) is a disease characterized by persistent airway inflammation and disordered macrophage function. The extent to which alterations in macrophage bioenergetics contribute to impaired antioxidant responses and disease pathogenesis has yet to be fully delineated. <b>Objectives:</b> Through the study of COPD alveolar macrophages (AMs) and peripheral monocyte-derived macrophages (MDMs), we sought to establish if intrinsic defects in core metabolic processes drive macrophage dysfunction and redox imbalance. <b>Methods:</b> AMs and MDMs from donors with COPD and healthy donors underwent functional, metabolic, and transcriptional profiling. <b>Measurements and Main Results:</b> We observed that AMs and MDMs from donors with COPD display a critical depletion in glycolytic- and mitochondrial respiration-derived energy reserves and an overreliance on glycolysis as a source for ATP, resulting in reduced energy status. Defects in oxidative metabolism extend to an impaired redox balance associated with defective expression of the NADPH-generating enzyme, ME1 (malic enzyme 1), a known target of the antioxidant transcription factor NRF2 (nuclear factor erythroid 2-related factor 2). Consequently, selective activation of NRF2 resets the COPD transcriptome, resulting in increased generation of TCA cycle intermediaries, improved energetic status, favorable redox balance, and recovery of macrophage function. <b>Conclusions:</b> In COPD, an inherent loss of metabolic plasticity leads to metabolic exhaustion and reduced redox capacity, which can be rescued by activation of the NRF2 pathway. Targeting these defects, via NRF2 augmentation, may therefore present an attractive therapeutic strategy for the treatment of the aberrant airway inflammation described in COPD.