Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with ...

New York University School of Medicine

The Chaim Sheba Medical Center

Endothelin-1 (ET-1) receptor antagonists increase plasma immunoreactive ET-1 levels. However, their effect on preproendothelin-1 (PPE-1) mRNA levels is still controversial. Few studies have found a decrease in PPE-1 mRNA levels in endothelial cells treated with the nonselective ETA/B receptor antagonist, and others demonstrated that an ETB blockade by the selective antagonist BQ788 increases PPE-1 mRNA levels. We studied the effect of ETA and ETB selective receptor antagonists on PPE-1 transcription, both in vitro and in vivo. Endothelial cells, transiently transfected with PPE-1 luciferase plasmid, were treated with ET-1 receptor antagonists. Bosentan, a dual ETA/B receptor antagonist, and BQ788 (ETB receptor antagonist) treatment resulted in a 1.6-fold and 1.3-fold increase, respectively in luciferase activity as compared with the untreated control. In contrast, the ETA receptor antagonist, BQ123, had no effect on luciferase activity. Transgenic mice that express the luciferase gene under the control of PPE-1 promoter were treated with Bosentan. Luciferase activity, PPE-1 mRNA levels, and plasma immunoreactive ET-1 levels were increased by 1.6-fold to 2.0-fold in the Bosentan-treated group compared with the untreated, control group. ET-1 receptor blockade increased PPE-1 transcription both in vitro and in vivo. The increased transcription can be attributed to ETB receptor blockade, because BQ-788, but not BQ-123, increased PPE-1 transcription.

Endothelin B receptor antagonist increases preproendothelin-1 transcription in bovine aortic endothelial cells and in vivo.

HIF-1 is a transcription factor that regulates genes involved in oxygen homeostasis. In normoxia, degradation of the HIF-1 alpha subunit is enabled by two prolyl hydroxylations at residues P402 and P564, while inactivation occurs through asparaginyl hydroxylation at residue N803 within its C-transactivation domain (C-TAD). For therapeutic angiogenesis purposes, HIF-1 alpha stabilization was previously achieved by either deleting its oxygen-dependent degradation domains, or introducing two proline point mutations at residues P402 and P564. We assessed the hypothesis that constitutive activation of HIF-1 alpha in addition to its stabilization would result in greater HIF-1 alpha transcriptional activity and angiogenic effects than mere stabilization of the molecule. For this, we constructed a Triple mutant HIF-1 alpha (TM), bearing mutations P402A and P564G N803A. Transient co-transfections with hypoxia-responsive element-luciferase construct revealed 2 to 2.5-fold increase in transcriptional activity of TM compared with P402A P564G double mutant and wild-type HIF-1 alpha. In-vitro angiogenesis assay using transfected human umbilical vein endothelial cells (HUVEC) showed that TM stimulated tube formation to a greater extent than both P402A P564G mutant and wild-type HIF-1 alpha. Accordingly, ELISA revealed that VEGF levels within the transfected HUVEC were about 10-fold greater with the TM.

Activation of C-transactivation domain is essential for optimal HIF-1 alpha-mediated transcriptional and angiogenic effects.

Hypoxia-inducible factor-1alpha (HIF-1alpha) is a key regulator of the response to low oxygen levels and has been used for therapeutic angiogenesis. Various routes of administration have been used for delivering genes to the ischemic region including the intramuscular (IM) and intraarterial routes. When compared with these delivery methods, the intravenous (IV) route confers many advantages, including less invasiveness and lower cost. However, its use is hampered by the fact that it does not result in specific and robust tissue expression of the genes. Our aim was to determine the feasibility, safety, and therapeutic efficacy of systemic administration of adenoviral-mediated HIF-1alpha targeted to the endothelium. Using confocal microscopy and biodistribution studies we demonstrated that a modified murine preproendothelin-1 promoter (PPE1-3x) can target gene expression specifically to endothelial cells within ischemic muscle following systemic IV administration in C57BL/6 mice. Accordingly, an adenovirus expressing a PPE1-3x-regulated stabilized HIF-1alpha molecule, further activated by constitutive activation of its C-transactivation domain (C-TAD), was created. Systemic tail-vein administration of this adenovirus in a mouse hindlimb ischemia model resulted in enhanced blood perfusion, improved clinical outcome, and increased capillary density without systemic toxicity, in contrast to the profound systemic side effects and lack of therapeutic efficacy following cytomegalovirus (CMV)-regulated HIF-1alpha administration. Collectively, these data suggest that transcriptionally controlled systemic proangiogenic gene therapy is feasible, safe, and efficacious.

Endothelial-targeted gene transfer of hypoxia-inducible factor-1alpha augments ischemic neovascularization following systemic administration.

Angiogenesis is an essential process for solid tumor development. To interfere with angiogenesis, AdPPE3x-E1, an adenovirus that is transcriptionally targeted to replicate in angiogenic endothelial cells, was constructed, by replacing the E1 promoter with the modified preproendothelin-1 promoter, PPE-1-3x, previously shown to induce specific transcription in angiogenic endothelial cells.

Systemic administration of a conditionally replicating adenovirus, targeted to angiogenesis, reduced lung metastases burden in cotton rats.

Transcription-targeted gene delivery directed against angiogenic endothelial cells is a new approach against advanced cancer. Moreover, the herpes simplex virus-thymidine kinase (HSV-TK) gene coupled with low dose radiotherapy is an efficient and externally controlled cytotoxic system. We have previously demonstrated enhanced endothelial-specific cell expression and killing using the modified murine pre-proendothelin-1 promoter (PPE1-3x) to direct adenoviral expression of a pro-apoptotic gene. The purpose of this study was to create an externally potentiated systemic antiangiogenic gene delivery system based on an adenoviral vector expressing HSV-TK under the regulation of PPE1-3X promoter combined with radiotherapy for eradicating metastatic cancer. Ad-PPE1-3x-TK induced endothelial-specific cell killing in-vitro upon introduction of the prodrug ganciclovir (GCV). BALB/c mice bearing a primary CT-26 colon carcinoma tumor showed tumor growth suppression and diminished tumor angiogenesis when the vector was administered intravenously, activated with GCV and potentiated with a single sub-therapeutic and non-toxic radiation dose. Moreover, intravenous administration of the vector, activated with GCV and potentiated with chest aimed radiation, to C57BL/6 mice bearing Lewis lung carcinoma metastases resulted in prolongation of mice survival. PPE1-3x-regulated HSV-TK expression was detected only in lung metastases in contrast to CMV-regulated expression. This novel system may benefit patients with metastatic disease.

Systemic administration of radiation-potentiated anti-angiogenic gene therapy against primary and metastatic cancer based on transcriptionally controlled HSV-TK.

Syndrome of inappropriate secretion of antidiuretic hormone and rhabdomyolysis complicating rhytidectomy.

Rhabdomyolysis is an uncommon complication of hyponatremia, reported previously only in case reports and small retrospective studies, and its underlying mechanism is controversial. Some studies support the hypothesis that the rapid correction of hyponatremia is responsible for rhabdomyolysis, whereas others emphasize the severity of the hyponatremia as a predisposing factor for rhabdomyolysis.

No association between hyponatremia and rhabdomyolysis in rats.

It is well recognized that macrophages in many contexts in vitro and in vivo display a spectrum of inflammatory features and functional properties. A convenient system to group together different subsets of macrophages has been the M1 (inflammatory)/M2 (anti-inflammatory) classification. In addition to other sites of inflammation, it is now established that atherosclerotic plaques contain both M1 and M2 macrophages. We review results made possible by a number of recent mouse models of atherosclerotic regression that, taken with other literature, have shown the M1/M2 balance in plaques to be dynamic, with M1 predominating in disease progression and M2 in regression. The regulation of the macrophage phenotype in plaques and the functional consequences of the M1 and M2 states in atherosclerosis will also be discussed.

Dynamic Aspects of Macrophage Polarization during Atherosclerosis Progression and Regression.

Programmed death-1 (PD-1) is an inhibitory co-receptor that is highly expressed in T lymphocytes that has been shown to downregulate inflammatory responses in several inflammatory diseases including systemic lupus erythematosus and rheumatoid arthritis. Yet, the role of PD-1 in psoriatic arthritis (PsA) has not been studied. In order to fill this gap, we measured the expression levels of PD-1 in peripheral T cells from patients with active disease. Twenty patients and fifteen age-matched healthy control subjects were recruited. The percentage of CD3(+)PD-1(+) T cells was measured by flow cytometry. Despite normal concentration of peripheral T cells, the expression levels of PD-1 were significantly higher in patients compared to healthy controls. Interestingly, among the patients, the expression levels inversely correlated with disease activity measured by disease activity scores (DAS28). PD-1 expression levels strongly correlated with the number of tender and swollen joints, but not with C-reactive protein (CRP) levels or psoriasis area and severity index (PASI). Functionally, in vitro ligation of PD-1 receptor in PsA T cells inhibited interleukin-2 (IL-2) secretion, Akt phosphorylation, and Rap1 activation. These findings suggest that PD-1 might serve as a biomarker for disease activity in PsA and highlight the need for additional studies in order to establish the role of PD-1 in PsA pathogenesis.

Michael Peled

Department of Medicine

New York University School of Medicine

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Michael Peled

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New York University School of Medicine

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Department of Medicine