Stromal cell-derived factor-1 alpha improves cardiac function in a novel diet-induced coronary atherosclerosis model, the SR-B1ΔCT/LDLR KO mouse.

BACKGROUND AND AIMS: There are a limited number of pharmacologic therapies for coronary artery disease, and few rodent models of occlusive coronary atherosclerosis and consequent myocardial infarction with which one can rapidly test new therapeutic approaches. Here, we characterize a novel, fertile, and easy-to-use HDL receptor (SR-B1)-based model of atherogenic diet-inducible, fatal coronary atherosclerosis, the SR-B1ΔCT/LDLR KO mouse. Additionally, we test intramyocardial injection of Stromal Cell-Derived Factor-1 alpha (SDF-1α), a potent angiogenic cytokine, as a possible therapy to rescue cardiac function in this mouse. METHODS: SR-B1ΔCT/LDLR KO mice were fed the Paigen diet or standard chow diet, and we determined the effects of the diets on cardiac function, histology, and survival. After two weeks of feeding either the Paigen diet (n = 24) or standard chow diet (n = 20), the mice received an intramyocardial injection of either SDF-1α or phosphate buffered saline (PBS). Cardiac function and angiogenesis were assessed two weeks later. RESULTS: When six-week-old mice were fed the Paigen diet, they began to die as early as 19 days later and 50% had died by 38 days. None of the mice maintained on the standard chow diet died by day 72. Hearts from mice on the Paigen diet showed evidence of cardiomegaly, myocardial infarction, and occlusive coronary artery disease. For the five mice that survived until day 28 that underwent an intramyocardial injection of PBS on day 15, the average ejection fraction (EF) decreased significantly from day 14 (the day before injection, 52.1 ± 4.3%) to day 28 (13 days after the injection, 30.6 ± 6.8%) (paired t-test, n = 5, p = 0.0008). Of the 11 mice fed the Paigen diet and injected with SDF-1α on day 15, 8 (72.7%) survived to day 28. The average EF for these 8 mice increased significantly from 48.2 ± 7.2% on day 14 to63.6 ± 6.9% on day 28 (Paired t-test, n = 8, p = 0.003). CONCLUSIONS: This new mouse model and treatment with the promising angiogenic cytokine SDF-1α may lead to new therapeutic approaches for ischemic heart disease.

Tumor Microenvironment Landscapes Supporting EGFR-mutant NSCLC Are Modulated at the Single-cell Interaction Level by Unesbulin Treatment.

Lung cancer is the leading cause of cancer deaths. Lethal pulmonary adenocarcinomas (ADC) present with frequent mutations in the EGFR. Genetically engineered murine models of lung cancer expedited comprehension of the molecular mechanisms driving tumorigenesis and drug response. Here, we systematically analyzed the evolution of tumor heterogeneity in the context of dynamic interactions occurring with the intermingled tumor microenvironment (TME) by high-resolution transcriptomics. Our effort identified vulnerable tumor-specific epithelial cells, as well as their cross-talk with niche components (endothelial cells, fibroblasts, and tumor-infiltrating immune cells), whose symbiotic interface shapes tumor aggressiveness and is almost completely abolished by treatment with Unesbulin, a tubulin binding agent that reduces B cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) activity. Simultaneous magnetic resonance imaging (MRI) analysis demonstrated decreased tumor growth, setting the stage for future investigations into the potential of novel therapeutic strategies for EGFR-mutant ADCs. SIGNIFICANCE: Targeting the TME is an attractive strategy for treatment of solid tumors. Here we revealed how EGFR-mutant landscapes are affected at the single-cell resolution level during Unesbulin treatment. This novel drug, by targeting cancer cells and their interactions with crucial TME components, could be envisioned for future therapeutic advancements.

Identification of a targetable KRAS-mutant epithelial population in non-small cell lung cancer.

Lung cancer is the leading cause of cancer deaths. Tumor heterogeneity, which hampers development of targeted therapies, was herein deconvoluted via single cell RNA sequencing in aggressive human adenocarcinomas (carrying Kras-mutations) and comparable murine model. We identified a tumor-specific, mutant-KRAS-associated subpopulation which is conserved in both human and murine lung cancer. We previously reported a key role for the oncogene BMI-1 in adenocarcinomas. We therefore investigated the effects of in vivo PTC596 treatment, which affects BMI-1 activity, in our murine model. Post-treatment, MRI analysis showed decreased tumor size, while single cell transcriptomics concomitantly detected near complete ablation of the mutant-KRAS-associated subpopulation, signifying the presence of a pharmacologically targetable, tumor-associated subpopulation. Our findings therefore hold promise for the development of a targeted therapy for KRAS-mutant adenocarcinomas.

Myeloid-Specific Deletion of Epsins 1 and 2 Reduces Atherosclerosis by Preventing LRP-1 Downregulation.

RATIONALE: Atherosclerosis is, in part, caused by immune and inflammatory cell infiltration into the vascular wall, leading to enhanced inflammation and lipid accumulation in the aortic endothelium. Understanding the molecular mechanisms underlying this disease is critical for the development of new therapies. Our recent studies demonstrate that epsins, a family of ubiquitin-binding endocytic adaptors, are critical regulators of atherogenicity. Given the fundamental contribution lesion macrophages make to fuel atherosclerosis, whether and how myeloid-specific epsins promote atherogenesis is an open and significant question. OBJECTIVE: We will determine the role of myeloid-specific epsins in regulating lesion macrophage function during atherosclerosis. METHODS AND RESULTS: We engineered myeloid cell-specific epsins double knockout mice (LysM-DKO) on an ApoE-/- background. On Western diet, these mice exhibited marked decrease in atherosclerotic lesion formation, diminished immune and inflammatory cell content in aortas, and reduced necrotic core content but increased smooth muscle cell content in aortic root sections. Epsins deficiency hindered foam cell formation and suppressed proinflammatory macrophage phenotype but increased efferocytosis and anti-inflammatory macrophage phenotype in primary macrophages. Mechanistically, we show that epsin loss specifically increased total and surface levels of LRP-1 (LDLR [low-density lipoprotein receptor]-related protein 1), an efferocytosis receptor with antiatherosclerotic properties. We further show that epsin and LRP-1 interact via epsin's ubiquitin-interacting motif domain. ox-LDL (oxidized LDL) treatment increased LRP-1 ubiquitination, subsequent binding to epsin, and its internalization from the cell surface, suggesting that epsins promote the ubiquitin-dependent internalization and downregulation of LRP-1. Crossing ApoE-/-/LysM-DKO mice onto an LRP-1 heterozygous background restored, in part, atherosclerosis, suggesting that epsin-mediated LRP-1 downregulation in macrophages plays a pivotal role in propelling atherogenesis. CONCLUSIONS: Myeloid epsins promote atherogenesis by facilitating proinflammatory macrophage recruitment and inhibiting efferocytosis in part by downregulating LRP-1, implicating that targeting epsins in macrophages may serve as a novel therapeutic strategy to treat atherosclerosis.

Fatty acid synthase mediates EGFR palmitoylation in EGFR mutated non-small cell lung cancer.

Metabolic reprogramming is widely known as a hallmark of cancer cells to allow adaptation of cells to sustain survival signals. In this report, we describe a novel oncogenic signaling pathway exclusively acting in mutated epidermal growth factor receptor (EGFR) non-small cell lung cancer (NSCLC) with acquired tyrosine kinase inhibitor (TKI) resistance. Mutated EGFR mediates TKI resistance through regulation of the fatty acid synthase (FASN), which produces 16-C saturated fatty acid palmitate. Our work shows that the persistent signaling by mutated EGFR in TKI-resistant tumor cells relies on EGFR palmitoylation and can be targeted by Orlistat, an FDA-approved anti-obesity drug. Inhibition of FASN with Orlistat induces EGFR ubiquitination and abrogates EGFR mutant signaling, and reduces tumor growths both in culture systems and in vivo Together, our data provide compelling evidence on the functional interrelationship between mutated EGFR and FASN and that the fatty acid metabolism pathway is a candidate target for acquired TKI-resistant EGFR mutant NSCLC patients.

Carboxy-terminal deletion of the HDL receptor reduces receptor levels in liver and steroidogenic tissues, induces hypercholesterolemia, and causes fatal heart disease.

The HDL receptor SR-BI mediates the transfer of cholesteryl esters from HDL to cells and controls HDL abundance and structure. Depending on the genetic background, loss of SR-BI causes hypercholesterolemia, anemia, reticulocytosis, splenomegaly, thrombocytopenia, female infertility, and fatal coronary heart disease (CHD). The carboxy terminus of SR-BI (505QEAKL509) must bind to the cytoplasmic adaptor PDZK1 for normal hepatic-but not steroidogenic cell-expression of SR-BI protein. To determine whether SR-BI's carboxy terminus is also required for normal protein levels in steroidogenic cells, we introduced into SR-BI's gene a 507Ala/STOP mutation that produces a truncated receptor (SR-BIΔCT). As expected, the dramatic reduction of hepatic receptor protein in SR-BIΔCT mice was similar to that in PDZK1 knockout (KO) mice. Unlike SR-BI KO females, SR-BIΔCT females were fertile. The severity of SR-BIΔCT mice's hypercholesterolemia was intermediate between those of SR-BI KO and PDZK1 KO mice. Substantially reduced levels of the receptor in adrenal cortical cells, ovarian cells, and testicular Leydig cells in SR-BIΔCT mice suggested that steroidogenic cells have an adaptor(s) functionally analogous to hepatic PDZK1. When SR-BIΔCT mice were crossed with apolipoprotein E KO mice (SR-BIΔCT/apoE KO), pathologies including hypercholesterolemia, macrocytic anemia, hepatic and splenic extramedullary hematopoiesis, massive splenomegaly, reticulocytosis, thrombocytopenia, and rapid-onset and fatal occlusive coronary arterial atherosclerosis and CHD (median age of death: 9 wk) were observed. These results provide new insights into the control of SR-BI in steroidogenic cells and establish SR-BIΔCT/apoE KO mice as a new animal model for the study of CHD.

Targeted BMI1 inhibition impairs tumor growth in lung adenocarcinomas with low CEBPα expression.

Lung cancer is the most common cause of cancer deaths. The expression of the transcription factor C/EBPα (CCAAT/enhancer binding protein α) is frequently lost in non-small cell lung cancer, but the mechanisms by which C/EBPα suppresses tumor formation are not fully understood. In addition, no pharmacological therapy is available to specifically target C/EBPα expression. We discovered a subset of pulmonary adenocarcinoma patients in whom negative/low C/EBPα expression and positive expression of the oncogenic protein BMI1 (B lymphoma Mo-MLV insertion region 1 homolog) have prognostic value. We also generated a lung-specific mouse model of C/EBPα deletion that develops lung adenocarcinomas, which are prevented by Bmi1 haploinsufficiency. BMI1 activity is required for both tumor initiation and maintenance in the C/EBPα-null background, and pharmacological inhibition of BMI1 exhibits antitumor effects in both murine and human adenocarcinoma lines. Overall, we show that C/EBPα is a tumor suppressor in lung cancer and that BMI1 is required for the oncogenic process downstream of C/EBPα loss. Therefore, anti-BMI1 pharmacological inhibition may offer a therapeutic benefit for lung cancer patients with low expression of C/EBPα and high BMI1.

Experimental Pilot Study of a Novel Endobronchial Drug Delivery Catheter.

BACKGROUND: An endobronchial infusion catheter introduced through a flexible bronchoscope channel has not been previously described. The aim of this study was to evaluate the technical feasibility of a new device. METHODS: Four porcine models underwent bronchoscopy with the infusion catheter. In the first experiment, methylene blue was injected into airway in volumes of 0.1, 0.3, or 1.0 mL into 2 animals. One animal was killed at 1 hour and the other at 24 hours after the procedure and gross dye diffusion was visually assessed. In the second experiment, a mixture of 80% sterile normal saline and 20% contrast media was injected into the airway in volumes of 0.3, 1.0, and 3.0 mL into 2 animals. One animal was killed at 7 days and the other at 20 days. Histologic evaluations were performed according to a bronchial damage scoring system. RESULTS: There was no perioperative morbidity. In the first experiment, infusion volumes of 0.1, 0.3, and 1.0 mL resulted in dye surrounding 67%±29%, 55%±17%, and 80%±20% of the infusion-site circumference, and longitudinal distribution of 4.0±1.7, 8.1±4.1, and 18.0±3.0 mm each, respectively. In the second experiment, infusion of 0.3 to 3.0 mL resulted in mild injury, inflammation, and hemorrhage/fibrin/thrombus at 7 and 20 days after surgery. CONCLUSIONS: Endobronchial infusion of dye and contrast media by the endobronchial drug delivery catheter showed that the media spread in a dose-dependent manner macroscopically and histologically. Further investigation will be required to assess the catheter as a new tool for localized drug delivery into the airway.

Success and failure rates of tumor genotyping techniques in routine pathological samples with non-small-cell lung cancer.

INTRODUCTION: Identification of some somatic molecular alterations in non-small-cell lung cancer (NSCLC) has become evidence-based practice. The success and failure rate of using commercially available tumor genotyping techniques in routine day-to-day NSCLC pathology samples is not well described. We sought to evaluate the success and failure rate of EGFR mutation, KRAS mutation, and ALK FISH in a cohort of lung cancers subjected to routine clinical tumor genotype. METHODS: Clinicopathologic data, tumor genotype success and failure rates were retrospectively compiled and analyzed from 381 patient-tumor samples. RESULTS: From these 381 patients with lung cancer, the mean age was 65 years, 61.2% were women, 75.9% were white, 27.8% were never smokers, 73.8% had advanced NSCLC and 86.1% had adenocarcinoma histology. The tumor tissue was obtained from surgical specimens in 48.8%, core needle biopsies in 17.9%, and as cell blocks from aspirates or fluid in 33.3% of cases. Anatomic sites for tissue collection included lung (49.3%), lymph nodes (22.3%), pleura (11.8%), bone (6.0%), brain (6.0%), among others. The overall success rate for EGFR mutation analysis was 94.2%, for KRAS mutation 91.6% and for ALK FISH 91.6%. The highest failure rates were observed when the tissue was obtained from image-guided percutaneous transthoracic core-needle biopsies (31.8%, 27.3%, and 35.3% for EGFR, KRAS, and ALK tests, respectively) and bone specimens (23.1%, 15.4%, and 23.1%, respectively). In specimens obtained from bone, the failure rates were significantly higher for biopsies than resection specimens (40% vs. 0%, p=0.024 for EGFR) and for decalcified compared to non-decalcified samples (60% vs. 5.5%, p=0.021 for EGFR). CONCLUSIONS: Tumor genotype techniques are feasible in most samples, outside small image-guided percutaneous transthoracic core-needle biopsies and bone samples from core biopsies with decalcification, and therefore expansion of routine tumor genotype into the care of patients with NSCLC may not require special tissue acquisition or manipulation.

Adequacy of lymph node transbronchial needle aspirates using convex probe endobronchial ultrasound for multiple tumor genotyping techniques in non-small-cell lung cancer.

INTRODUCTION: Adequate tumor acquisition is essential to identify somatic molecular alterations in non-small-cell lung cancer (NSCLC), such as epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) translocations. The success and failure rates for tumor genotyping of tissue obtained from fine-needle aspirates of nodal tissue using a convex probe endobronchial ultrasound (CP-EBUS) and other diagnostic modalities in routine NSCLC care have not been described. METHODS: Clinicopathologic data, tumor genotype success and failure rates were retrospectively compiled and analyzed from 207 patient-tumor samples sent for routine tumor genotype in clinical practice, including 42 patient-tumor samples obtained from hilar or mediastinal lymph nodes using CP-EBUS. RESULTS: The median age of the patients was 65 years, 62.3% were women, 77.8% were white, 26.6% were never smokers, 73.9% had advanced NSCLC, and 84.1% had adenocarcinoma histology. Tumor tissue was obtained from CP-EBUS-derived hilar or mediastinal nodes in 42 cases (20.2% of total). In this latter cohort, the overall success rate for EGFR mutation analysis was 95.2%, for Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation 90.5%, and for ALK fluorescence in situ hybridization 90.5%. In the complete 207 tumors, the success rate for EGFR was 92.3%, for KRAS 91.8%, and for ALK 89.9%. The failure rates were not significantly different when comparing CP-EBUS-derived nodal tissue versus all other samples or versus surgical biopsies of mediastinal nodes, but were significantly lower than image-guided percutaneous transthoracic core-needle biopsies. CONCLUSIONS: The success rate of multiple tumor genomic analyses techniques for EGFR, KRAS, and ALK gene abnormalities using routine lung cancer tissue samples obtained from hilar or mediastinal lymph nodes by means of CP-EBUS exceeds 90%, and this method of tissue acquisition is not inferior to other specimen types. Tumor genotype techniques are feasible in most CP-EBUS-derived samples and therefore further expansion of routine tumor genotype for the care of patients with NSCLC may be possible using targeted sample acquisition through CP-EBUS.

Molecular biomarkers of vascular dysfunction in obstructive sleep apnea.

Untreated and long-lasting obstructive sleep apnea (OSA) may lead to important vascular abnormalities, including endothelial cell (EC) dysfunction, hypertension, and atherosclerosis. We observed a correlation between microcirculatory reactivity and endothelium-dependent release of nitric oxide in OSA patients. Therefore, we hypothesized that OSA affects (micro)vasculature and we aimed to identify vascular gene targets of OSA that could possibly serve as reliable biomarkers of severity of the disease and possibly of vascular risk. Using quantitative RT-PCR, we evaluated gene expression in skin biopsies of OSA patients, mouse aortas from animals exposed to 4-week intermittent hypoxia (IH; rapid oscillations in oxygen desaturation and reoxygenation), and human dermal microvascular (HMVEC) and coronary artery endothelial cells (HCAEC) cultured under IH. We demonstrate a significant upregulation of endothelial nitric oxide synthase (eNOS), tumor necrosis factor-alpha-induced protein 3 (TNFAIP3; A20), hypoxia-inducible factor 1 alpha (HIF-1α?? and vascular endothelial growth factor (VEGF) expression in skin biopsies obtained from OSA patients with severe nocturnal hypoxemia (nadir saturated oxygen levels [SaO2]<75%) compared to mildly hypoxemic OSA patients (SaO2 75%-90%) and a significant upregulation of vascular cell adhesion molecule 1 (VCAM-1) expression compared to control subjects. Gene expression profile in aortas of mice exposed to IH demonstrated a significant upregulation of eNOS and VEGF. In an in vitro model of OSA, IH increased expression of A20 and decreased eNOS and HIF-1α expression in HMVEC, while increased A20, VCAM-1 and HIF-1αexpression in HCAEC, indicating that EC in culture originating from distinct vascular beds respond differently to IH stress. We conclude that gene expression profiles in skin of OSA patients may correlate with disease severity and, if validated by further studies, could possibly predict vascular risk in OSA patients.

Smoking status and self-reported race affect the frequency of clinically relevant oncogenic alterations in non-small-cell lung cancers at a United States-based academic medical practice.

INTRODUCTION: The identification of somatic genomic aberrations in non-small-cell lung cancer (NSCLC) is part of evidence-based practice guidelines for care of patients with NSCLC. We sought to establish the frequency and correlates with these changes in routine patient-tumor sample pairs. METHODS: Clinicopathologic data and tumor genotype were retrospectively compiled and analyzed from an overall cohort of 381 patient-tumor samples. RESULTS: Of these patients, 75.9% self-reported White race, 13.1% Asian, 6.5% Black, 27.8% were never-smokers, 54.9% former-smokers and 17.3% current-smokers. The frequency of EGFR mutations was 23.9% (86/359), KRAS mutations 34.2% (71/207) and ALK FISH positivity 9.1% (23/252) in tumor samples, and almost all had mutually exclusive results for these oncogenes. In tumors from White, Black and Asian patients, the frequencies of EGFR mutations were 18.4%, 18.2% and 62%, respectively; of ALK FISH positivity 7.81%, 0% and 14.8%, respectively; and of KRAS mutations 41.6%, 20% and 0%. These patterns changed significant with increasing pack-year history of smoking. In White patients, the frequencies of EGFR mutations and ALK FISH positivity decreased with increasing pack-year cohorts; while the frequencies of KRAS mutations increased. Interestingly, in Asian patients the frequencies of EGFR mutations were similar in never smokers and in the cohorts with less than 45pack-year histories of smoking and only decreased in the 45pack-year plus cohort. CONCLUSIONS: The frequencies of somatic EGFR, KRAS, and ALK gene abnormalities using routine lung cancer tissue samples from our United States-based academic medical practice reflect the diverse ethnicity (with a higher frequency of EGFR mutations in Asian patients) and smoking patterns (with an inverse correlation between EGFR mutation and ALK rearrangement) of our tested population. These results may help other medical practices appreciate the expected results from introduction of routine tumor genotyping techniques into their day-to-day care of NSCLC.

Challenges in using cultured primary rodent hepatocytes or cell lines to study hepatic HDL receptor SR-BI regulation by its cytoplasmic adaptor PDZK1.

BACKGROUND: PDZK1 is a four PDZ-domain containing cytoplasmic protein that binds to a variety of membrane proteins via their C-termini and can influence the abundance, localization and/or function of its target proteins. One of these targets in hepatocytes in vivo is the HDL receptor SR-BI. Normal hepatic expression of SR-BI protein requires PDZK1 - <5% of normal hepatic SR-BI is seen in the livers of PDZK1 knockout mice. Progress has been made in identifying features of PDZK1 required to control hepatic SR-BI in vivo using hepatic expression of wild-type and mutant forms of PDZK1 in wild-type and PDZK1 KO transgenic mice. Such in vivo studies are time consuming and expensive, and cannot readily be used to explore many features of the underlying molecular and cellular mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: Here we have explored the potential to use either primary rodent hepatocytes in culture using 2D collagen gels with newly developed optimized conditions or PDZK1/SR-BI co-transfected cultured cell lines (COS, HEK293) for such studies. SR-BI and PDZK1 protein and mRNA expression levels fell rapidly in primary hepatocyte cultures, indicating this system does not adequately mimic hepatocytes in vivo for analysis of the PDZK1 dependence of SR-BI. Although PDZK1 did alter SR-BI protein expression in the cell lines, its influence was independent of SR-BI's C-terminus, and thus is not likely to occur via the same mechanism as that which occurs in hepatocytes in vivo. CONCLUSIONS/SIGNIFICANCE: Caution must be exercised in using primary hepatocytes or cultured cell lines when studying the mechanism underlying the regulation of hepatic SR-BI by PDZK1. It may be possible to use SR-BI and PDZK1 expression as sensitive markers for the in vivo-like state of hepatocytes to further improve primary hepatocyte cell culture conditions.

Noncanonical role of the PDZ4 domain of the adaptor protein PDZK1 in the regulation of the hepatic high density lipoprotein receptor scavenger receptor class B, type I (SR-BI).

The four PDZ (PDZ1 to PDZ4) domain-containing adaptor protein PDZK1 controls the expression, localization, and function of the HDL receptor scavenger receptor class B, type I (SR-BI), in hepatocytes in vivo. This control depends on both the PDZ4 domain and the binding of SR-BI's cytoplasmic C terminus to the canonical peptide-binding sites of either the PDZ1 or PDZ3 domain (no binding to PDZ2 or PDZ4). Using transgenic mice expressing in the liver domain deletion (ΔPDZ2 or ΔPDZ3), domain replacement (PDZ2→1), or target peptide binding-negative (PDZ4(G389P)) mutants of PDZK1, we found that neither PDZ2 nor PDZ3 nor the canonical target peptide binding activity of PDZ4 were necessary for hepatic SR-BI regulatory activity. Immunohistochemical studies established that the localization of PDZK1 on hepatocyte cell surface membranes in vivo is dependent on its PDZ4 domain and the presence of SR-BI. Analytical ultracentrifugation and hydrogen deuterium exchange mass spectrometry suggested that the requirement of PDZ4 for localization and SR-BI regulation is not due to PDZ4-mediated oligomerization or induction of conformational changes in the PDZ123 portion of PDZK1. However, surface plasmon resonance analysis showed that PDZ4, but not the other PDZ domains, can bind vesicles that mimic the plasma membrane. Thus, PDZ4 may potentiate PDZK1's regulation of SR-BI by promoting its lipid-mediated attachment to the cytoplasmic membrane. Our results show that not all of the PDZ domains of a multi-PDZ domain-containing adaptor protein are required for its biological activities and that both canonical target peptide binding and noncanonical (peptide binding-independent) capacities of PDZ domains may be employed by a single such adaptor for optimal in vivo activity.

Molecular analysis of the prostacyclin receptor's interaction with the PDZ1 domain of its adaptor protein PDZK1.

The prostanoid prostacyclin, or prostaglandin I2, plays an essential role in many aspects of cardiovascular disease. The actions of prostacyclin are mainly mediated through its activation of the prostacyclin receptor or, in short, the IP. In recent studies, the cytoplasmic carboxy-terminal domain of the IP was shown to bind several PDZ domains of the multi-PDZ adaptor PDZK1. The interaction between the two proteins was found to enhance cell surface expression of the IP and to be functionally important in promoting prostacyclin-induced endothelial cell migration and angiogenesis. To investigate the interaction of the IP with the first PDZ domain (PDZ1) of PDZK1, we generated a nine residue peptide (KK(411)IAACSLC(417)) containing the seven carboxy-terminal amino acids of the IP and measured its binding affinity to a recombinant protein corresponding to PDZ1 by isothermal titration calorimetry. We determined that the IP interacts with PDZ1 with a binding affinity of 8.2 µM. Using the same technique, we also determined that the farnesylated form of carboxy-terminus of the IP does not bind to PDZ1. To understand the molecular basis of these findings, we solved the high resolution crystal structure of PDZ1 bound to a 7-residue peptide derived from the carboxy-terminus of the non-farnesylated form of IP ((411)IAACSLC(417)). Analysis of the structure demonstrates a critical role for the three carboxy-terminal amino acids in establishing a strong interaction with PDZ1 and explains the inability of the farnesylated form of IP to interact with the PDZ1 domain of PDZK1 at least in vitro.

RhoB differentially controls Akt function in tumor cells and stromal endothelial cells during breast tumorigenesis.

Tumors are composed of cancer cells but also a larger number of diverse stromal cells in the tumor microenvironment. Stromal cells provide essential supports to tumor pathophysiology but the distinct characteristics of their signaling networks are not usually considered in developing drugs to target tumors. This oversight potentially confounds proof-of-concept studies and increases drug development risks. Here, we show in established murine and human models of breast cancer how differential regulation of Akt by the small GTPase RhoB in cancer cells or stromal endothelial cells determines their dormancy versus outgrowth when angiogenesis becomes critical. In cancer cells in vitro or in vivo, RhoB functions as a tumor suppressor that restricts EGF receptor (EGFR) cell surface occupancy as well as Akt signaling. However, after activation of the angiogenic switch, RhoB functions as a tumor promoter by sustaining endothelial Akt signaling, growth, and survival of stromal endothelial cells that mediate tumor neoangiogenesis. Altogether, the positive impact of RhoB on angiogenesis and progression supercedes its negative impact in cancer cells themselves. Our findings elucidate the dominant positive role of RhoB in cancer. More generally, they illustrate how differential gene function effects on signaling pathways in the tumor stromal component can complicate the challenge of developing therapeutics to target cancer pathophysiology.

In vivo generation of transplantable human hematopoietic cells from induced pluripotent stem cells.

Lineage-restricted cells can be reprogrammed to a pluripotent state known as induced pluripotent stem (iPS) cells through overexpression of 4 transcription factors. iPS cells are similar to human embryonic stem (hES) cells and have the same ability to generate all the cells of the human body, including blood cells. However, this process is extremely inefficient and to date has been unsuccessful at differentiating iPS into hematopoietic stem cells (HSCs). We hypothesized that iPS cells, injected into NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ immunocompromised (NSG) mice could give rise to hematopoietic stem/progenitor cells (HSPCs) during teratoma formation. Here, we report a novel in vivo system in which human iPS cells differentiate within teratomas to derive functional myeloid and lymphoid cells. Similarly, HSPCs can be isolated from teratoma parenchyma and reconstitute a human immune system when transplanted into immunodeficient mice. Our data provide evidence that in vivo generation of patient customized cells is feasible, providing materials that could be useful for transplantation, human antibody generation, and drug screening applications.

Temporal network based analysis of cell specific vein graft transcriptome defines key pathways and hub genes in implantation injury.

Vein graft failure occurs between 1 and 6 months after implantation due to obstructive intimal hyperplasia, related in part to implantation injury. The cell-specific and temporal response of the transcriptome to vein graft implantation injury was determined by transcriptional profiling of laser capture microdissected endothelial cells (EC) and medial smooth muscle cells (SMC) from canine vein grafts, 2 hours (H) to 30 days (D) following surgery. Our results demonstrate a robust genomic response beginning at 2 H, peaking at 12-24 H, declining by 7 D, and resolving by 30 D. Gene ontology and pathway analyses of differentially expressed genes indicated that implantation injury affects inflammatory and immune responses, apoptosis, mitosis, and extracellular matrix reorganization in both cell types. Through backpropagation an integrated network was built, starting with genes differentially expressed at 30 D, followed by adding upstream interactive genes from each prior time-point. This identified significant enrichment of IL-6, IL-8, NF-κB, dendritic cell maturation, glucocorticoid receptor, and Triggering Receptor Expressed on Myeloid Cells (TREM-1) signaling, as well as PPARα activation pathways in graft EC and SMC. Interactive network-based analyses identified IL-6, IL-8, IL-1α, and Insulin Receptor (INSR) as focus hub genes within these pathways. Real-time PCR was used for the validation of two of these genes: IL-6 and IL-8, in addition to Collagen 11A1 (COL11A1), a cornerstone of the backpropagation. In conclusion, these results establish causality relationships clarifying the pathogenesis of vein graft implantation injury, and identifying novel targets for its prevention.

Extrahepatic high-density lipoprotein receptor SR-BI and apoA-I protect against deep vein thrombosis in mice.

OBJECTIVE: Deep vein thrombosis (DVT) and pulmonary embolism are frequent causes of morbidity and mortality. The goal of our study was to determine whether plasma high-density lipoprotein (HDL), which inversely correlates with the risk of cardiovascular events, affects DVT. METHODS AND RESULTS: Using a murine DVT model of inferior vena cava stenosis, we demonstrated that deficiency of the HDL receptor, scavenger receptor class B type I (SR-BI), promotes venous thrombosis. As SR-BI(-/-) mice have increased plasma cholesterol levels and abnormal HDL particles, we tested SR-BI(-/-) mice with an SR-BI liver transgene that normalizes both parameters. These mice also exhibited increased susceptibility to DVT, indicating a protective role of extrahepatic SR-BI. Mice lacking the major HDL apolipoprotein apoA-I or endothelial nitric oxide synthase (eNOS) (a downstream target of endothelial SR-BI signaling) also had a prothrombotic phenotype. Intravenous infusion of human apoA-I, an HDL component and SR-BI ligend, prevented DVT in wild-type but not SR-BI(-/-) or eNOS(-/-) mice, suggesting that its effect is mediated by SR-BI and eNOS. Intravenous apoA-I infusion abolished histamine-induced platelet-endothelial interactions, which are important for DVT initiation. CONCLUSIONS: An apoA-I (HDL)-SR-BI-eNOS axis is highly protective in DVT and may provide new targets for prophylaxis and treatment of venous thrombosis.