Extracellular Hsp90 mediates an NF-κB dependent inflammatory stromal program: implications for the prostate tumor microenvironment.

BACKGROUND: The tumor microenvironment (TME) plays an essential role in supporting and promoting tumor growth and progression. An inflammatory stroma is a widespread hallmark of the prostate TME, and prostate tumors are known to co-evolve with their reactive stroma. Cancer-associated fibroblasts (CAFs) within the reactive stroma play a salient role in secreting cytokines that contribute to this inflammatory TME. Although a number of inflammatory mediators have been identified, a clear understanding of key factors initiating the formation of reactive stroma is lacking. METHODS: We explored whether tumor secreted extracellular Hsp90 alpha (eHsp90α) may initiate a reactive stroma. Prostate stromal fibroblasts (PrSFs) were exposed to exogenous Hsp90α protein, or to conditioned medium (CM) from eHsp90α-expressing prostate cancer cells, and evaluated for signaling, motility, and expression of prototypic reactive markers. In tandem, ELISA assays were utilized to characterize Hsp90α-mediated secreted factors. RESULTS: We report that exposure of PrSFs to eHsp90 upregulates the transcription and protein secretion of IL-6 and IL-8, key inflammatory cytokines known to play a causative role in prostate cancer progression. Cytokine secretion was regulated in part via a MEK/ERK and NF-κB dependent pathway. Secreted eHsp90α also promoted the rapid and durable activation of the oncogenic inflammatory mediator signal transducer and activator of transcription (STAT3). Finally, eHsp90 induced the expression of MMP-3, a well-known mediator of fibrosis and the myofibroblast phenotype. CONCLUSIONS: Our results provide compelling support for eHsp90α as a transducer of signaling events culminating in an inflammatory and reactive stroma, thereby conferring properties associated with prostate cancer progression.

Interleukin-1 beta induces differential gene expression in aortic smooth muscle cells.

PURPOSE: Abdominal aortic aneurysms are characterized by an accelerated turnover of extracellular matrix proteins and by an inflammatory infiltrate that releases the cytokines interleukin-1 beta and tumor necrosis factor-alpha. We examined the gene expression of human aneurysmal aortic smooth muscle cells and normal aortic smooth muscle cells after treatment with interleukin-1 beta and tumor necrosis factor-alpha by measuring the changes in smooth muscle cell collagen, elastin, collagenase, and tissue inhibitor of metalloproteinase messenger ribonucleic acid levels in response to these cytokines. METHODS: Biopsy of aneurysmal aorta (n = 6) and donor normal aorta (n = 3) was obtained at operation. Medial smooth muscle cells were cultured, passaged (P2 to P4), and incubated with 0, 10, 100, or 1000 pg/ml interleukin-1 beta, tumor necrosis factor-alpha, or platelet-derived growth factor for 24 hours. Total ribonucleic acid was harvested. Percentage changes in messenger ribonucleic acid from control levels for type I and type III procollagen, elastin, collagenase, 72 kDa type IV collagenase, tissue inhibitor of metalloproteinase-1, and tissue inhibitor of metalloproteinase-2 were measured by Northern hybridization. Analyses were performed with analysis of variance (p < 0.05). All comparisons between aneurysmal aortic smooth muscle cells and normal aortic smooth muscle cells represent comparisons between one aneurysmal aorta and one normal aorta. RESULTS: Added interleukin-1 beta resulted in significant, dose-dependent increases in the collagenase messenger ribonucleic acid level at all concentrations tested in both aneurysmal aorta and normal aorta. The increase in the collagenase messenger ribonucleic acid level ranged from a minimum increase of 123% for 10 pg/ml interleukin-1 beta in aneurysmal aortic smooth muscle cells to a maximum of 450% for 1000 pg/ml interleukin-1 beta in normal aortic smooth muscle cells. Interleukin-1 beta caused a significant decrease in the steady-state messenger ribonucleic acid levels for type 1 procollagen in both aneurysmal and normal aorta. The greatest reduction in type 1 procollagen messenger ribonucleic acid levels occurred at 100 pg/ml interleukin-1 beta in both aneurysmal aortic smooth muscle cells (-39%) and normal aortic smooth muscle cells (-48%). The only observed qualitative difference between aneurysmal aortic smooth muscle cells and normal aortic smooth muscle cells was the change in tissue inhibitor of metalloproteinase-1 messenger ribonucleic acid levels in response to added interleukin-1 beta. In aneurysmal aortic smooth muscle cells interleukin-1 beta at 1000 pg/ml significantly increased messenger ribonucleic acid levels by 82%, whereas levels of tissue inhibitor of metalloproteinase-1 messenger ribonucleic acid in normal aortic smooth muscle cells did not change in response to added interleukin-1 beta. Interleukin-1 beta did not alter messenger ribonucleic acid levels for type III procollagen, elastin, type IV collagenase, or tissue inhibitor of metalloproteinase-2 in aneurysmal aorta or normal aorta. When tumor necrosis factor-alpha or platelet-derived growth factor were added, this did not significantly change aneurysmal aortic smooth muscle cells messenger ribonucleic acid levels for collagenase, type IV collagenase, tissue inhibitor of metalloproteinase-1, tissue inhibitor of metalloproteinase-2, and type I and type III procollagen. CONCLUSIONS: These findings suggest that interleukin-1 beta, through its effect on smooth muscle cell collagenase and collagen gene expression, mediates the increased matrix turnover observed in aneurysms. Macrophages may induce changes in aortic smooth muscle cell gene expression in a paracrine manner that could lead to aneurysm formation.

Femorofemoral bypass for aortofemoral graft limb occlusion: a ten-year experience.

PURPOSE: Aortofemoral bypass (AFB) is a durable reconstruction; however, graft limb occlusion occurs in 10% to 20% of patients and results in limb ischemia. Treatment of AFB limb occlusion has been debated, but many recommended femorofemoral bypass (FFB). FFB grafts have had excellent patency rates. The durability of FFB specifically for AFB limb occlusion has not been reported. This study retrospectively examined a 10-year experience with FFB for AFB limb occlusion to determine FFB performance. METHODS: Between 1982 and 1992, FFB was performed on occluded AFB limbs in 22 patients (14 men and 8 women). Reoperation was performed for disabling claudication in five cases, but the remaining 17 patients (77%) had critical limb ischemia. FFB originated from the contralateral patent AFB limb in all cases. Distal anastomosis was to the common femoral artery (n = 8) or the profunda femoris (n = 14). FFB graft patency was confirmed by direct Doppler arterial examination over a mean follow-up of 47 months. RESULTS: The cumulative life-table primary patency rate of FFB was 54% at 5 years. Reoperative procedures performed in nine cases resulted in a secondary patency rate of 84% at 5 years. The limb salvage rate was also 84% at 5 years, reflecting the impact of successful reoperation. Major amputations (two below-knee, one above-knee) were necessary in only three cases. There were no perioperative deaths after FFB, and the cumulative 5-year survival rate was 77%. CONCLUSION: Aortic graft limb occlusion occurs less frequently than failure of infrainguinal grafts making the success of specific reoperative strategies difficult to document reliably. This study suggests that FFB is a safe and durable alternative for AFB limb failure. An aggressive policy of reoperation has resulted in successful extension of FFB graft function and an excellent rate of limb salvage.

Endothelial cells can synthesize leukotriene B4.

Leukotriene B4 (LTB4) from vascular endothelium may play a key role in the genesis of atherosclerotic lesions. However, the ability of this tissue to synthesize LTB4 is controversial. To resolve this issue arachidonic acid metabolism was characterized in cultures of confluent monolayers of a rabbit aortic endothelial cell line by use of both high-pressure liquid chromatography and radioimmunoassay. Cells were grown to confluence in Dulbecco's modified Eagle's medium/Ham's F12 with 5% fetal bovine serum. Lipoxygenase activity was studied by placing the cells in Hank's balanced salt solution with 2 mumol/L indomethacin. After 30 minutes preincubation with indomethacin cells were exposed to either arachidonic acid (10 mumol/L) or arachidonic acid labeled with radioactive carbon (14C) (1 microCi; SA 58 mCi/mmol) and then stimulated with 9.5 mumol/L calcium ionophore A23187 for 55 minutes. Studies of the cyclooxygenase activity were performed without preincubating with indomethacin. Samples were prepared for high-pressure liquid chromatography by evaporation to dryness under a vacuum and resuspending in 2 ml of 1:1 methanol/water. Tritium-labeled standards were added before loading the 14C-labeled samples on the column. Radiolabeled arachidonic acid metabolites were separated by high-pressure liquid chromatography and detected by means of a dual channel flow-through radiodetector that monitors both 14C and 3H. Based on coelution with authentic standards three lipoxygenase metabolites of arachidonic acid have been identified: LTB4, 12- and 5-hydroxyeicosatetraneoic acid. Leukotriene B4 was further characterized by ultraviolet spectral analysis and inhibition studies with use of nordihydroguaiaretic acid. Quantitation was facilitated by commercially available radioimmunoassay kits. An average of 600 pg LTB4/10(6) cells was measured from separate experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Treatment of established prosthetic vascular graft infection with antibiotics preferentially concentrated in leukocytes.

The efficacy of treating established vascular graft infections with rifampin and clindamycin (preferentially concentrated in leukocytes) and cefazolin (not concentrated in leukocytes) was studied in a canine model. Infrarenal aortic, 6 mm by 6 cm knitted Dacron double velour grafts were implanted and infected with 10(8) colony-forming units (CFU) of coagulase-positive Staphyloccus aureus organisms injected intravenously immediately after graft placement. Antibiotic therapy was instituted at 3 months postimplantation. Three groups were studied: (I) untreated controls (n = 3); (II) therapy with intravenous cefazolin 15 mg/kg/8 hr for 28 days (n = 7); and (III) combined therapy with intravenous rifampin 13 mg/kg/24 hr and intravenous clindamycin 13 mg/kg/8 hr for 28 days (n = 7). Grafts were removed for quantitative bacteriologic studies after the 28-day course of therapy. Two group I control grafts remained patent with 6.4 X 10(6) and 8.1 X 10(3) CFU S. aureus/gm of graft. The third control graft was thrombosed. Two group II animals demonstrated 1.6 X 10(7) and 2.3 X 10(5) CFU S. aureus organisms/gram of graft, respectively; the remaining five group II grafts were free of organisms. All group III grafts were sterile--a significant difference (p less than 0.05) from group I grafts. In this experimental model, established prosthetic graft infections were eradicated by intensive treatment with antibiotics preferentially concentrated in leukocytes.