Citations

Abstract Tissue injury triggers reparative processes that often involve endothelial progenitor cells (EPCs) recruitment. We hypothesized that atherosclerotic renal artery stenosis (ARAS) activates homing signals that would be detectable in both the kidney and EPCs, and attenuated on renal repair using selective cell-based therapy. Pigs were treated with intrarenal autologous EPC after 6 weeks of ARAS. Four weeks later, expression of homing-related signals in EPC and kidney, single kidney function, microvascular (MV) density, and morphology were compared with untreated ARAS and normal control pigs (n = 7 each). Compared with normal EPC, EPC from ARAS pigs showed increased stromal cell-derived factor (SDF)-1, angiopoietin-1, Tie-2, and c-kit expression, but downregulation of erythropoietin (EPO) and its receptor. The ARAS kidney released the c-kit-ligand stem cell factor, uric acid, and EPO, and upregulated integrin β2, suggesting activation of corresponding homing signaling. However, angiopoietin-1 and SDF-1/CXCR4 were not elevated. Administration of EPC into the stenotic kidney restored angiogenic activity, improved MV density, renal hemodynamics and function, decreased fibrosis and oxidative stress, and attenuated endogenous injury signals. The ARAS kidney releases specific homing signals corresponding to cognate receptors expressed by EPC. EPC show plasticity for organ-specific recruitment strategies, which are upregulated in early atherosclerosis. EPC are renoprotective as they attenuated renal dysfunction and damage in chronic ARAS, and consequently decreased the injury signals. Importantly, manipulation of homing signals may potentially allow therapeutic opportunities to increase endogenous EPC recruitment.

Objective— Hypertension and hypercholesterolemia might interfere with renal repair mechanisms. We hypothesized that simvastatin improves the survival of endothelial progenitor cells (EPC) in the renal microenvironment imposed by concurrent renovascular hypertension and dietary hypercholesterolemia (HTC). Methods and Results— Pigs were studied after 12 weeks of no intervention (n=6), HTC (n=6), or HTC+ oral simvastatin supplementation (80 mg/day, n=5). EPC were also isolated and studied in vitro after exposure to the proapoptotic oxidized low-density lipoprotein with or without coincubation with simvastatin. Renal hemodynamics, function, and endothelial function were evaluated in vivo, and the number of CD34+/KDR+ EPC, apoptosis, oxidative stress, inflammation, and fibrosis in renal tissue studied ex vivo. Compared with normal kidney, the HTC kidney showed endothelial dysfunction and increased oxidative stress, interstitial macrophage filtration, and fibrosis. The number of EPC in the kidney increased, as did their apoptosis (0.85±0.24% versus 0.22±0.07%, P <0.05 versus normal). Simvastatin did not affect blood pressure, cholesterol levels, basal renal function, or number of renal EPC in HTC, but it improved endothelial function; blunted renal oxidative stress, inflammation, and fibrosis; and attenuated EPC apoptosis (to 0.37±0.09%, P <0.05 versus HTC). Simvastatin also significantly decreased oxidized low-density lipoprotein–induced EPC apoptosis in vitro. Conclusion— EPC are recruited but undergo apoptosis in the HTC kidney, likely because of a hostile microenvironment. Simvastatin rescues renal repair mechanisms in HTC and counteracts renal damage, which may account for its protective effects on the kidney during exposure to cardiovascular risk factors.

AbstractA paucity of validated kinase targets in human multiple myeloma has delayed clinical deployment of kinase inhibitors in treatment strategies. We therefore conducted a kinome-wide small interfering RNA (siRNA) lethality study in myeloma tumor lines bearing common t(4;14), t(14;16), and t(11;14) translocations to identify critically vulnerable kinases in myeloma tumor cells without regard to preconceived mechanistic notions. Fifteen kinases were repeatedly vulnerable in myeloma cells, including AKT1, AK3L1, AURKA, AURKB, CDC2L1, CDK5R2, FES, FLT4, GAK, GRK6, HK1, PKN1, PLK1, SMG1, and TNK2. Whereas several kinases (PLK1, HK1) were equally vulnerable in epithelial cells, others and particularly G protein–coupled receptor kinase, GRK6, appeared selectively vulnerable in myeloma. GRK6 inhibition was lethal to 6 of 7 myeloma tumor lines but was tolerated in 7 of 7 human cell lines. GRK6 exhibits lymphoid-restricted expression, and from coimmunoprecipitation studies we demonstrate that expression in myeloma cells is regulated via direct association with the heat shock protein 90 (HSP90) chaperone. GRK6 silencing causes suppression of signal transducer and activator of transcription 3 (STAT3) phosphorylation associated with reduction in MCL1 levels and phosphorylation, illustrating a potent mechanism for the cytotoxicity of GRK6 inhibition in multiple myeloma (MM) tumor cells. As mice that lack GRK6 are healthy, inhibition of GRK6 represents a uniquely targeted novel therapeutic strategy in human multiple myeloma.

Hacs1, a SH3 and SAM domain‐containing adaptor protein, is up‐regulated by IL‐4 in activated B cells and strongly expressed in dendritic cells. To elucidate the function of Hacs1 in immune regulation, we generated Hacs1 − / − mice by deletion of the SH3 and SAM domains. Hacs1 − / − mice were viable and fertile and had normal bone marrow B‐cell development and normal splenic T‐ and B‐cell populations. However, adult Hacs1 − / − mice had increased peritoneal Bla cells (IgM + CD5 + ). On immunization with T‐cell‐independent antigen TNP‐Ficoll, Hacs1 − / − mice had increased production of anti‐TNP IgM and IgG3. Purified splenic B cells from Hacs1 − / − mice showed increased cell proliferation on BCR (B‐cell receptor) stimulation. We further demonstrate that the Hacs1 − / − B cells had increased global tyrosine phosphorylation, including tyrosine kinases Lyn and Akt. Both T‐helper type 1 (T h 1) and T‐helper type 2 (T h 2) humoral responses were enhanced in Hacs1 − / − mice. In vitro bone marrow‐derived Hacs1 − / − dendritic cells showed increased IL‐12 production on stimulation with ovalbumin (OVA). This study suggests that Hacs1 is an immunoinhibitory adaptor that might be a useful target for immune suppression therapy.—Wang, D., Stewart, A. K., Zhuang, L., Zhu, Y., Wang, Y., Shi, C., Keating, A., Slutsky, A., Zhang, H., Wen, X.‐Y. Enhanced adaptive immunity in mice lacking the immunoinhibitory adaptor Hacs1. FASEBJ. 24, 947–956 (20l0). www.fasebj.org

Abstract Activating epidermal growth factor receptor (EGFR) mutations are common in many cancers including glioblastoma. However, clinical responses to EGFR inhibitors are infrequent and short-lived. We show that the Src family kinases (SFK) Fyn and Src are effectors of oncogenic EGFR signaling, enhancing invasion and tumor cell survival in vivo. Expression of a constitutively active EGFR mutant, EGFRvIII, resulted in activating phosphorylation and physical association with Src and Fyn, promoting tumor growth and motility. Gene silencing of Fyn and Src limited EGFR- and EGFRvIII-dependent tumor cell motility. The SFK inhibitor dasatinib inhibited invasion, promoted tumor regression, and induced apoptosis in vivo, significantly prolonging survival of an orthotopic glioblastoma model expressing endogenous EGFRvIII. Dasatinib enhanced the efficacy of an anti-EGFR monoclonal antibody (mAb 806) in vivo, further limiting tumor growth and extending survival. Examination of a large cohort of clinical samples showed frequent coactivation of EGFR and SFKs in glioblastoma patients. These results establish a mechanism linking EGFR signaling with Fyn and Src activation to promote tumor progression and invasion in vivo and provide rationale for combined anti-EGFR and anti-SFK targeted therapies. [Cancer Res 2009;69(17):6889–98]

Background— Monocyte chemoattractant proteins (MCPs) play an important role in mediating inflammatory processes. Hypertension (HTN) is associated with inflammation as well as impaired cardiac microcirculatory function and structure, but the contribution of MCPs to these alterations remained unclear. This study tested the hypothesis that MCPs regulate cardiac microvascular function and structure in experimental HTN. Methods and Results— Pigs (n=6 per group) were studied after 10 weeks of normal, renovascular HTN, or renovascular HTN+ bindarit (MCPs inhibitor, 50 mg/kg/d PO). Left ventricular (LV) function, myocardial microvascular permeability, and fractional vascular volume were assessed by fast computed tomography before and after adenosine infusion (400 μg/kg/min). Myocardial fibrosis, inflammation, and microvascular remodeling were determined ex vivo. Hypertension was not altered by bindarit, but LV hypertrophy and diastolic function were improved. In response to adenosine, myocardial microvascular permeability increased in HTN (from 0.0083±0.0009 to 0.0103±0.0011 AU, P =0.038 versus baseline) and fractional vascular volume decreased, whereas both remained unchanged in normal and HTN+bindarit pigs. HTN upregulated endothelin-1 expression, myocardial inflammation, and microvascular wall thickening, which were inhibited by bindarit. Conclusions— MCPs partly mediate myocardial inflammation, fibrosis, vascular remodeling, and impaired vascular integrity induced by hypertension. Inhibition of MCPs could potentially be a therapeutic target in hypertensive cardiomyopathy.

Background— Endothelial progenitor cells (EPCs) promote neovascularization and endothelial repair. Renal artery stenosis (RAS) may impair renal function by inducing intrarenal microvascular injury and remodeling. We investigated whether replenishment with EPCs would protect the renal microcirculation in chronic experimental renovascular disease. Methods and Results— Single-kidney hemodynamics and function were assessed with the use of multidetector computed tomography in vivo in pigs with RAS, pigs with RAS 4 weeks after intrarenal infusion of autologous EPCs, and controls. Renal microvascular remodeling and angiogenic pathways were investigated ex vivo with the use of micro–computed tomography, histology, and Western blotting. EPCs increased renal expression of angiogenic factors, stimulated proliferation and maturation of new vessels, and attenuated renal microvascular remodeling and fibrosis in RAS. Furthermore, EPCs normalized the blunted renal microvascular and filtration function. Conclusions— The present study shows that a single intrarenal infusion of autologous EPCs preserved microvascular architecture and function and decreased microvascular remodeling in experimental chronic RAS. It is likely that restoration of the angiogenic cascade by autologous EPCs involved not only generation of new vessels but also acceleration of their maturation and stabilization. This contributed to preserving the blood supply, hemodynamics, and function of the RAS kidney, supporting EPCs as a promising therapeutic intervention for preserving the kidney in renovascular disease.

Abstract B7-H4 is an immunoglobulin superfamily molecule and shown to be inhibitory for T-cell responses. To explore physiologic roles of B7-H4, we created B7-H4–deficient (KO) mice by genetic targeting. B7-H4KO mice are healthy and their T- and B-cell responses to polyclonal antigens are in normal range. However, B7-H4KO mice are more resistant to infection by Listeria monocytogenes than their littermates. Within 3 days after infection, bacterial colonies in livers and spleens are significantly lower than the controls, suggesting a role of B7-H4 in enhancing innate immunity. Further studies demonstrate that neutrophils increase in peripheral organs of B7-H4KO mice more so than their littermates but their bactericidal functions remain unchanged. Augmented innate resistance is completely dependent on neutrophils, even in the absence of adaptive immunity. In vitro B7-H4 inhibits the growth of bone marrow–derived neutrophil progenitors, suggesting an inhibitory function of B7-H4 in neutrophil expansion. Our results identify B7-H4 as a negative regulator of the neutrophil response to infection and provide a new target for manipulation of innate immunity.

AbstractAs multiple myeloma tumors universally dysregulate cyclin D genes we conducted high-throughput chemical library screens for compounds that induce suppression of cyclin D2 promoter transcription. The top-ranked compound was a natural triterpenoid, pristimerin. Strikingly, the early transcriptional response of cells treated with pristimerin closely resembles cellular responses elicited by proteosome inhibitors, with rapid induction of heat shock proteins, activating transcription factor 3 (ATF3), and CHOP. Enzymatic assays and immunoblotting confirm that pristimerin rapidly (< 90 minutes) and specifically inhibits chymotrypsin-like proteosome activity at low concentrations (< 100 nM) and causes accumulation of cellular ubiquitinated proteins. Notably, cytotoxic triterpenoids including pristimerin inhibit NF-κB activation via inhibition of IKKα or IKKβ, whereas proteosome inhibitors instead suppress NF-κB function by impairing degradation of ubiquitinated IκB. By inhibiting both IKK and the proteosome, pristimerin causes overt suppression of constitutive NF-κB activity in myeloma cells that may mediate its suppression of cyclin D. Multiple myeloma is exquisitely sensitive to proteosome or NF-κB pathway inhibition. Consistent with this, pristimerin is potently and selectively lethal to primary myeloma cells (IC50 < 100 nM), inhibits xenografted plasmacytoma tumors in mice, and is synergistically cytotoxic with bortezomib—providing the rationale for pharmaceutical development of triterpenoid dual-function proteosome/NF-κB inhibitors as therapeutics for human multiple myeloma and related malignancies.

Purpose To assess the effectiveness of depot octreotide for the prevention of diarrhea during pelvic radiation therapy. Patients and Methods Patients receiving pelvic radiation therapy (planned minimum dose, 45 Gy; 1.7 to 2.1 Gy daily) were eligible for the study. From May 10, 2002, through October 14, 2005, 125 patients were randomly allocated in a double-blind fashion to receive octreotide (100 μg, administered subcutaneously on day 1, followed by depot octreotide, 20 mg, administered intramuscularly on days 2 and 29; n = 62) or to receive a placebo (n = 63). Results Grade 0, 1, 2, and 3 diarrhea were observed in 18%, 31%, 31%, and 21% of patients in the octreotide arm, respectively, and in 25%, 32%, 22%, and 21% of patients in the placebo arm, respectively (P = .64). Grade 0, 1, 2, and 3 abdominal cramps were observed in 32%, 45%, 21%, and 2% of patients receiving octreotide, respectively, and in 51%, 24%, 21%, and 5% of patients receiving the placebo, respectively (P = .053). Some patient-reported symptoms were worse in the octreotide group, including nocturnal bowel movements (70% v 45%; P = .004), clustering of bowel movements (90% v 69%; P = .004), and bleeding with bowel movements (57% v 35%; P = .01). Conclusion As administered in this study, octreotide did not decrease diarrhea during pelvic radiation therapy. Some gastrointestinal symptoms were worse in patients treated with octreotide. Octreotide is not indicated for prevention of diarrhea during pelvic radiation therapy.