Michael K. Dame

Perfluorocarbon protects lung epithelial cells from neutrophil-mediated injury in an in vitro model of liquid ventilation therapy

Liquid ventilation with perfluorocarbon has been effective in improving gas exchange and pulmonary function in the setting of acute respiratory failure. How improvement is brought about remains unknown. In the present study, we examined perfluorocarbon for effects on neutrophil function and for effects on neutrophil-epithelial cell interactions that could underlie its in vivo activity. Exposure of neutro-phils in vitro to perfluorocarbon followed by washing did not interfere with their ability to generate oxidants or release proteolytic enzymes upon subsequent stimulation. Likewise, such treatment did not interfere with subsequent adhesion of the neutrophils to monolayers of epithelial cells or with neutrophil-induced injury to these cells. In contrast, when perfluorocarbon was added to neutrophils and epithelial cells together and the neutrophils then stimulated, it reduced their adhesive interaction with the target cells and concomitantly reduced target cell injury. In companion studies, cells were obtained by bronchial lavage of perfluorocar-bon-treated patients with acute respiratory distress syndrome (ARDS) and analyzed for oxidant production. Oxidant-generating capacity by the cells obtained from the bronchial lavage fluid was similar to that of peripheral blood neutrophils. These data are consistent with the suggestion that perfluorocarbon protects cells in vitro from neutrophil-mediated injury, at least in part, by acting as a mechanical barrier. These findings are consistent with a potential mechanism of action in vivo.

Cyclic stretching of mesangial cells up-regulates intercellular adhesion molecule-1 and leukocyte adherence: a possible new mechanism for glomerulosclerosis.

Intraglomerular hypertension is a primary causal factor in the progressive glomerulosclerosis that characterizes diabetic nephropathy or severe renal ablation. However, inflammation of the glomerular mesangium also participates in at least the early phase of these diseases. In glomerulonephritis, where inflammation is thought to be the predominant causal factor, intraglomerular hypertension is also often present. Mesangial cells (MCs) are critical in orchestrating key functions of the glomerulus including extracellular matrix metabolism, cytokine production, and interaction with leukocytes. Because MCs are subject to increased stretching when intraglomerular hypertension is present, and in glomerulonephritis MC/leukocyte interactions seem to be mediated primarily via the up-regulation of intercellular adhesion molecule-1 (ICAM-1), we examine the possibility that cyclic stretching is a stimulus for increased MC ICAM-1 activity. We demonstrate that the normal low levels of MC ICAM-1 mRNA and protein are dramatically up-regulated by even short intervals of cyclic stretch. This effect is dose- and time-dependent, and requires little amplitude and a brief period of elongation for significant induction. Stretch-induced MC ICAM-1 also leads to a marked elevation in phagocytic leukocyte adherence. This stimulated adherence is equal or greater than that induced by the inflammatory cytokine tumor necrosis factor-alpha, whereas an additive effect occurs when both are applied in combination. Our results indicate that stretch-induced ICAM-1 may provide a direct link between hypertension and inflammation in the progression of injury and glomerulosclerosis in diabetes, renal ablation, and other forms of glomerulonephritis.

Tumor-selective proteotoxicity of verteporfin inhibits colon cancer progression independently of YAP1

In an oxygen- and nutrient-deprived environment, verteporfin kills colon cancer cells by inducing proteotoxicity. Aggregates kill cancer cells The drug verteporfin is used clinically to enhance phototherapy and may also inhibit the transcription factor YAP1, which is often active in cancers. However, Zhang et al. found a different path to toxicity for verteporfin-mediated death of colorectal cancer cells. Verteporfin triggered the accumulation of toxic amounts of protein oligomers that selectively killed colorectal cancer cells in mice and in cells cultured under hypoxic and nutrient-deprived conditions. Normal cells in culture and in tumor-adjacent tissue sections from mice cleared these aggregates through autophagy and survived. Thus, verteporfin produces tumor-selective proteotoxicity, which may be a useful therapeutic for patients with solid tumors. Yes-associated protein 1 (YAP1) is a transcriptional coactivator in the Hippo signaling pathway. Increased YAP1 activity promotes the growth of tumors, including that of colorectal cancer (CRC). Verteporfin, a drug that enhances phototherapy to treat neovascular macular degeneration, is an inhibitor of YAP1. We found that verteporfin inhibited tumor growth independently of its effects on YAP1 or the related protein TAZ in genetically or chemically induced mouse models of CRC, in patient-derived xenografts, and in enteroid models of CRC. Instead, verteporfin exhibited in vivo selectivity for killing tumor cells in part by impairing the global clearance of high–molecular weight oligomerized proteins, particularly p62 (a sequestrome involved in autophagy) and STAT3 (signal transducer and activator of transcription 3; a transcription factor). Verteporfin inhibited cytokine-induced STAT3 activity and cell proliferation and reduced the viability of cultured CRC cells. Although verteporfin accumulated to a greater extent in normal cells than in tumor cells in vivo, experiments with cultured cells indicated that the normal cells efficiently cleared verteporfin-induced protein oligomers through autophagic and proteasomal pathways. Culturing CRC cells under hypoxic or nutrient-deprived conditions (modeling a typical CRC microenvironment) impaired the clearance of protein oligomers and resulted in cell death, whereas culturing cells under normoxic or glucose-replete conditions protected cell viability and proliferation in the presence of verteporfin. Furthermore, verteporfin suppressed the proliferation of other cancer cell lines even in the absence of YAP1, suggesting that verteporfin may be effective against multiple types of solid cancers.

Endothelial cell determinants of susceptibility to neutrophil-mediated killing.

Vascular endothelial cell injury plays an important role in the pathogenesis of inflammatory-mediated tissue injury. In the current study, we assessed injury in primary cultures of endothelial cells obtained from different sites within the same species, comparing rat dermal microvascular and rat lung microvascular endothelial cells. Dermal microvascular-derived endothelial cells were more sensitive to killing by PMA (phorbol myristate acetate)-activated human neutrophils than were endothelial cells derived from lung microvasculature. Lung endothelial cells stimulated with interferon-gamma plus lipopolysaccharide (IFNgamma + LPS) generated high levels of nitric oxide (*NO), while dermal endothelial cells stimulated with IFNgamma + LPS generated significantly lower levels of *NO. Under conditions of *NO generation (IFNgamma + LPS stimulation), or in the presence of the *NO donor, S-nitroso-N-acetyl penicillamine (SNAP), endothelial cell killing by PMA-activated neutrophils was reduced. Lung endothelial cells stimulated with PMA generated less superoxide (02*-) than dermal endothelial cells. Under conditions of *NO generation (IFNgamma + LPS stimulation), or in the presence of SNAP, O2*- release from endothelial cells was reduced. Endothelial cell-derived *NO appeared to play a significant role in attenuating the neutrophil-mediated killing. The differences in the ability of endothelial cells to generate *NO and 02*- underlies, at least in part, the differences in susceptibility of these cells to injury by activated neutrophils.

Substrate-dependent differences in growth and biological properties of fibroblasts and epithelial cells grown in microcarrier culture

Normal diploid human fibroblasts and first passage monkey kidney epithelial cells were examined for growth and metabolic activity on microcarriers made from glass and on microcarriers made from DEAE-dextran. The cells grew to a higher density (cells cm2 of surface area) on the glass microcarriers made from glass and on microcarriers made from DEAE-dextran. The cells grew to a higher density (cells/cm2 of surface area) on the glass microcarriers than they did on the DEAE-dextran microcarriers and morphological differences were observed between the cells growing on the two substrates. On the DEAE-dextran microcarriers, the cells were much more resistant to protease-mediated detachment than were the cells on the glass microcarriers. In these respects, the cells grown on the glass microcarriers were similar to cells grown in conventional monolayer culture. Interestingly, the cells grown on the DEAE-dextran microcarriers expressed higher levels of proteolytic enzyme activity than the cells grown on the glass microcarriers. Substrate-dependent differences in prostaglandin production also occurred--both in unstimulated cells and in cells stimulated with 12-0-tetradecanoyl phorbol acetate. The unstimulated cells on the glass microcarriers produced slightly higher levels of three different prostaglandins than did the cells on the DEAE-dextran microcarriers. However, after stimulation the levels were much higher in the DEAE-dextran microcarrier cultures than in the glass microcarrier cultures. In contrast to these results, there was no significant, substrate-dependent difference in the production of infectious herpes simplex virus. Taken together, these findings suggest that when commercially-useful cells such as normal fibroblasts and epithelial cells are grown in large quantities on microcarriers, the nature of the substrate may have a profound effect on the growth and physiology of the cells. They also suggest that when microcarriers are used, unexpected results based on preliminary work in conventional monolayer culture may be obtained.

Induction of matrix metalloproteinase-1 (MMP-1) during epidermal invasion of the stroma in human skin organ culture: keratinocyte stimulation of fibroblast MMP-1 production

Organ cultures of human skin were incubated for 8 days under growth factor-free conditions or exposed to 10 ng ml–1 of human recombinant epidermal growth factor (EGF) during the incubation period. Normal histological features were preserved in the absence of growth factor, while epithelial cells underwent a proliferative response and invaded the underlying stroma in the presence of exogenous EGF. The same concentrations of EGF that induced stromal invasion also resulted in up-regulation of matrix metalloproteinase-9 (MMP-9; 92-kD gelatinase B) in organ culture and keratinocyte monolayer culture, and expression of MMP-1 (interstitial collagenase) in organ culture and fibroblast monolayer culture. When skin organ cultures were exposed to a potent, irreversible EGF–receptor tyrosine kinase (EGF–RTK) antagonist along with EGF, abnormal histological features were reversed, and MMP-9 production was suppressed. In contrast, EGF-RKT antagonism had only a modest inhibitory effect on MMP-1 production. Culture fluid from keratinocytes grown in monolayer culture stimulated fibroblast proliferation and MMP-1 elaboration. Treatment of fibroblasts with the same EGF–RTK antagonist inhibited keratinocyte-induced fibroblast proliferation but had only a modest inhibitory effect (approximately 20% inhibition) on MMP-1 production. In contrast, treatment of dermal fibroblasts with Interleukin-1 Receptor Antagonist had no effect on keratinocyte-induced fibroblast growth but strongly inhibited MMP-1 production (greater than 70% inhibition). These data indicate that stromal invasion by epithelial cells in EGF-treated skin is associated with events occurring in both the epidermis and dermis. The direct effect of the exogenous growth factor appears to be primarily on the epidermis. Dermal events reflect, at least in part, a response to factors elaborated in the epidermis.

Fibroblast Response to Gadolinium: Role for Platelet-Derived Growth Factor Receptor

Objective:The purpose of this study was to assess the effects of gadolinium (Gd3+), provided as gadolinium chloride, on fibroblast function. Materials and Methods:Human dermal fibroblasts in monolayer culture and intact skin in organ culture were exposed to the lanthanide metal (1–20 &mgr;m). Results:Increased proliferation was observed, in association with upregulation of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinases-1, without an apparent increase in production of type I procollagen. A platelet-derived growth factor (PDGF) receptor-blocking antibody inhibited fibroblast proliferation in response to Gd3+ as did inhibitors of signaling pathways—that is, mitogen-activated protein kinase and phosphatidylinositol-3 kinase pathways—that are activated by PDGF. Conclusion:The responses to gadolinium chloride are similar to responses previously seen with chelated Gd3+ in clinically used magnetic resonance imaging contrast agents. Fibroblast responses appear to reflect Gd3+-induced PDGF receptor activation and downstream signaling. Increased dermal fibroblast proliferation in conjunction with effects on matrix metalloproteinase-1 and tissue inhibitor of metalloproteinases-1 could contribute to the fibroplastic/fibrotic changes seen in the lesional skin of individuals with nephrogenic systemic fibrosis.

Regulation of collagen turnover in human skin fibroblasts exposed to a gadolinium-based contrast agent.

Objective:Nephrogenic systemic fibrosis is a clinical syndrome linked with exposure in renal failure patients to gadolinium-based contrast agents (GBCAs) during magnetic resonance imaging. Recently, we demonstrated that GBCA exposure led to increased matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinases-1 (TIMP-1) levels in human skin fibroblasts. The goals of the present work were to assess the relationship between altered MMP-1/TIMP-1 expression and collagen production/deposition, and the intracellular signaling events that lead from GBCA stimulation to altered MMP-1 and TIMP-1 production. Materials and Methods:Human dermal fibroblasts were treated with one of the currently used GBCAs (Omniscan). Proliferation was quantified as were levels of MMP-1, TIMP-1, procollagen type I, and collagen type I. Signaling events were concomitantly assessed, and signaling inhibitors were used. Results:Fibroblasts exposed to Omniscan had increases in both MMP-1 and TIMP-1 levels. Omniscan treatment interfered with collagen turnover, leading to increased type I collagen deposition without an increase in type I procollagen production. U0126, an inhibitor of mitogen-activated protein kinase signaling, and LY294002, a phosphatidylinositol-3 kinase inhibitor, reduced MMP-1 levels. U0126 also reduced TIMP-1 levels, but LY294002 increased TIMP-1. Conclusion:These data provide evidence for complex regulation of collagen deposition in Omniscan-treated skin. They suggest that the major effect of Omniscan exposure is on an enzyme/inhibitor system that regulates collagen breakdown rather than on collagen production, per se.

Matrix metalloproteinases (MMPs) in fresh human prostate tumour tissue and organ-cultured prostate tissue: Levels of collagenolytic and gelatinolytic MMPs are low, variable and different in fresh tissue versus organ-cultured tissue

Prostate tissue was obtained from 22 radical prostatectomies (performed for clinical management of prostate carcinoma) immediately after surgery. A small piece of tissue was fixed immediately in formalin and used for routine histology while a second piece was frozen in OCT and used for immuno-histochemistry. Another small piece was used for isolation of epithelial and stromal cells. The remainder of the tissue was cut into 2 × 2 mm pieces and incubated in organ culture for 8 days. In organ culture, non-malignant, basal epithelial cells underwent a proliferative response. This was accompanied by de-differentiation of glandular structures and by migration of epithelial cells across the surface of the tissue. Erosion of the basement membrane could also be seen in places, but was not widespread. Invasion of epithelial cells into the adjacent stroma was not evident. Production of matrix metalloproteinases (MMPs) with gelatinolytic activity or collagenolytic activity was assessed in organ culture and compared to expression patterns in fresh tissue. MMP-1 (interstitial collagenase) and MMP-9 (92-kDa gelatinase B) were undetectable or low in fresh tissue specimens. Both enzymes were detected in organ culture and both increased over time. Even after 6 days, however, there was only a low level of gelatin-hydrolytic activity and no measurable collagen-hydrolytic activity. In past studies we used organ cultures of normal skin and malignant skin tumours (basal cell carcinomas) to help elucidate the role of collagenolytic and gelatinolytic MMPs in epithelial cell invasion (Varani et al, 2000). Compared to MMP levels observed in skin, levels of these enzymes in prostate are low. The low level of collagenolytic and gelatinolytic MMPs in fresh prostate tissue and in organ-cultured prostate tissue may help explain why there is little tissue destruction in many primary prostate tumours and why the majority of such tumours remain confined to the prostate for extended periods.

Responses of Human Skin in Organ Culture and Human Skin Fibroblasts to a Gadolinium-Based MRI Contrast Agent: Comparison of Skin from Patients with End-Stage Renal Disease and Skin from Healthy Subjects

Objective:Nephrogenic systemic fibrosis is a clinical syndrome occurring in a small subset of patients with end-stage renal disease (ESRD). Exposure to certain of the gadolinium-based contrast agents during magnetic resonance imaging appears to be a trigger. The pathogenesis of the disease is largely unknown. The present study addresses potential pathophysiologic mechanisms. Materials and Methods:We have compared responses in organ-cultured skin and skin fibroblasts from individuals with ESRD to responses of healthy control subjects to Omniscan treatment. Results:Treatment of skin from ESRD patients with Omniscan stimulated production of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinases-1, but not type I procollagen. The same treatment also stimulated an increase in hyaluronan production. Similar results were seen with skin from normal controls but basal levels were higher in ESRD patients. Fibroblasts in monolayer culture gave the same responses, but there were no differences based on whether the cells were isolated from the skin of healthy subjects or those with ESRD. Conclusion:These data indicate that Omniscan exposure alters an enzyme/inhibitor system responsible for regulating collagen turnover in the skin and directly stimulates hyaluronan production. The higher basal levels of type I procollagen, matrix metalloproteinase-1, tissue inhibitor of metalloproteinases-1, and hyaluronan in the skin from ESRD patients could contribute to the sensitivity of this patient population to fibrotic changes, which might be induced by exposure to some of the gadolinium-based contrast agents.