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We have previously demonstrated the effectiveness of adenovirus-mediated expression of antisense urokinase-type plasminogen activator receptor (uPAR) and matrix metalloproteinase-9 (MMP-9) in inhibiting tumor invasion in vitro and ex vivo. However, the therapeutic effect of the adenovirus-mediated antisense approach was shown to be transient and required potentially toxic, high viral doses. In contrast, RNA interference (RNAi)-mediated gene targeting may be superior to the traditional antisense approach, because the target mRNA is completely degraded and the molar ratio of siRNA required to degrade the target mRNA is very low. Here, we have examined the siRNA-mediated target RNA degradation of uPAR and MMP-9 in human glioma cell lines. Using RNAi directed toward uPAR and MMP-9, we achieved specific inhibition of uPAR and MMP-9. This bicistronic construct (pUM) inhibited the formation of capillary-like structures in both in vitro and in vivo models of angiogenesis. We demonstrated that blocking the expression of these genes results in significant inhibition of glioma tumor invasion in Matrigel and spheroid invasion assay models. RNAi for uPAR and MMP-9 inhibited cell proliferation, and significantly reduced the levels of phosphorylated forms of MAPK, ERK, and AKT signaling pathway molecules when compared with parental and empty vector/scrambled vector-transfected SNB19 cells. Furthermore, using RNAi to simultaneously target two proteases resulted in total regression of pre-established intracerebral tumor growth. Our results provide evidence that the use of hairpin siRNA expression vectors for uPAR and MMP-9 may provide an effective tool for cancer therapy.

To determine the relationship between increased triggering receptor expressed on myeloid cells (TREM)-1 and plaque stability in atherosclerotic carotid stenosis.The mRNA transcripts and protein for TREM-1, MMP-1, MMP-9, collagen type I (COL1A1) and collagen type III (COL3A1) were analyzed by qPCR and immunofluorescence in both tissues and VSMCs isolated from atherosclerotic carotid plaques of symptomatic and asymptomatic patients with carotid stenosis.The TREM-1, MMP-1 and MMP-9 mRNA transcripts were significantly increased (TREM-1, p 0.01; MMP-1, p 0.01 and MMP-9, p 0.001) while COL1A1 and COL3A1 mRNA transcripts were decreased (p 0.001) in VSMCs isolated from carotid plaques of symptomatic (S) than asymptomatic (AS) patients. Stimulation of cells with TNF-α further increased the mRNA transcripts of TREM-1, MMPs, COL1A1 and COL3A1. Modulation of TREM-1 by treatment with TREM-1 decoy receptor rTREM-1/Fc, and either TREM-1 antibodies or TREM-1 siRNA attenuated the TNF-α-induced expression of MMP-1 and MMP-9 (p 0.01) and COL1A1 and COL3A1 (p 0.01) in S compared to AS VSMCs isolated from carotid plaques. Inhibition of NF-kB (BAY 11-7085), JNK (SP600125) and PI3K (LY294002) signaling pathways decreased the expression of TREM-1 (p 0.01), MMP-1 (p 0.001) and MMP-9 (p 0.01) in TNF-α-treated VSMCs isolated from S carotid plaques compared to AS patients.Increased expression of TREM-1 in S compared to AS patients involving MMP-1 and MMP-9 suggest a potential role of TREM-1 in plaque destabilization. Selective blockade of TREM-1 may contribute to the development of new therapies and promising targets for stabilizing vulnerable atherosclerotic plaques.

Although degradation of extracellular matrix by matrix metalloproteinases (MMPs) is thought to be involved in symptomatic (S) carotid plaques in atherosclerosis, the mechanisms of MMP expression are poorly understood. Here, we demonstrate that collagen loss in vascular smooth vessel cells (VSMCs) isolated from S plaques was induced by epidermal growth factor (EGF) through the activation of p38-MAPK and JNK-MAPK pathways. Inhibitors of p38-MAPK and JNK-MAPK signaling pathways downregulated the expression of MMP-1 and MMP-9. In addition, we examined whether v-ets erythroblastosis virus E26 oncogene homologue 1 (Ets-1), an important regulator of different genes, is involved in destabilizing S plaques in patients with carotid stenosis. We demonstrate that EGF induces Ets-1 expression and decreases interstitial and basement membrane collagen in vascular smooth muscle cells (VSMCs) from patients with carotid stenosis. Increased expression of MMP-1 and -9 and decreased collagen mRNA transcripts were also found in Ets-1-overexpressed VSMCs. Transfection with both dominant-negative form of Ets-1 and small interfering RNA blocked EGF-induced MMP-1 and -9 expressions and increased the mRNA transcripts for collagen I (α1) and collagen III (α1) in S compared with asymptomatic (AS) carotid plaques. Inhibitors of p38-MAPK (SB202190) and JNK-MAPK (SP600125) signaling pathways decreased the expression of Ets-1, MMP-1, and MMP-9 and increased collagen type I and III expression in EGF-treated VSMCs. This study provides a mechanistic insight into the role of Ets-1 in the plaque destabilization in patients with carotid stenosis involving p38-MAPK and JNK signaling pathways.

Solar ultraviolet (UV) radiation can cause severe damage to the skin and is the primary cause of most skin cancer. UV radiation causes DNA damage leading to mutations and also activates the Erbb2/HER2 receptor through indirect mechanisms involving reactive oxygen species. We hypothesized that Erbb2 activation accelerates the malignant progression of UV-induced skin cancer. Following the induction of benign squamous papillomas by UV exposure of v-ras(Ha) transgenic Tg.AC mice, mice were treated topically with the Erbb2 inhibitor AG825 and tumor progression monitored. AG825 treatment reduced tumor volume, increased tumor regression, and delayed the development of malignant squamous cell carcinoma (SCC). Progression to malignancy was associated with increased Erbb2 and ADAM12 (A Disintegin And Metalloproteinase 12) transcripts and protein, while inhibition of Erbb2 blocked the increase in ADAM12 message upon malignant progression. Similarly, human SCC and SCC cell lines had increased ADAM12 protein and transcripts when compared to normal controls. To determine whether Erbb2 up-regulation of ADAM12 contributed to malignant progression of skin cancer, Erbb2 expression was modulated in cultured SCC cells using forced over-expression or siRNA targeting, demonstrating up-regulation of ADAM12 by Erbb2. Furthermore, ADAM12 transfection or siRNA targeting revealed that ADAM12 increased both the migration and invasion of cutaneous SCC cells. Collectively, these results suggest Erbb2 up-regulation of ADAM12 as a novel mechanism contributing to the malignant progression of UV-induced skin cancer. Inhibition of Erbb2/HER2 reduced tumor burden, increased tumor regression, and delayed the progression of benign skin tumors to malignant SCC in UV-exposed mice. Inhibition of Erbb2 suppressed the increase in metalloproteinase ADAM12 expression in skin tumors, which in turn increased migration and tumor cell invasiveness.

Atherosclerosis is a chronic inflammatory disease with atherosclerotic plaques containing inflammatory cells, including T-lymphocytes, dendritic cells (DCs) and macrophages that are responsible for progression and destabilization of atherosclerotic plaques. Stressed cells undergoing necrosis release molecules that act as endogenous danger signals to alert and activate innate immune cells. In atherosclerotic tissue the number of DCs increases with the progression of the lesion and produce several inflammatory cytokines and growth factors. Triggering receptor expressed on myeloid cells (TREM)-1 plays a crucial role in inflammation. However, relationship of DCs and the role of TREM-1 with the stability of atherosclerotic plaques have not been examined. In this study, we investigated the heterogeneity of the plaque DCs, myeloid (mDC1 and mDC2) and plasmacytoid (pDCs), and examined the expression of TREM-1 and their co-localization with DCs in the plaques from symptomatic (S) and asymptomatic (AS) patients with carotid stenosis. We found increased expression of HLA-DR, fascin, and TREM-1 and decreased expression of TREM-2 and α-smooth muscle actin in S compared to AS atherosclerotic carotid plaques. Both TREM-1 and fascin were co-localized suggesting increased expression of TREM-1 in plaque DCs of S compared to AS patients. These data were supported by increased mRNA transcripts of TREM-1 and decreased mRNA transcripts of TREM-2 in carotid plaques of S compared to AS patients. There was higher density of both CD1c+ mDC1 and CD141+ mDC2 in the carotid plaques from AS compared to S patients, where as the density of CD303+ pDCs were higher in the carotid plaques of S compared to AS patients. These findings suggest a potential role of pDCs and TREM-1 in atherosclerotic plaque vulnerability. Thus, newer therapies could be developed to selectively block TREM-1 for stabilizing atherosclerotic plaques.

Increased activation of epidermal growth factor receptor (EGFR) family members such as HER2/Erbb2 can result in more aggressive disease, resistance to chemotherapy and reduced survival of head and neck squamous cell carcinoma (HNSCC) patients. In order to identify mechanisms through which these receptor tyrosine kinases accelerate tumor progression, the regulation of metalloprotease expression by EGFR family members was investigated in 11 squamous cell carcinoma (SCC) cell lines. HER2 expression was significantly correlated with ADAM12 (A Disintegrin And Metalloprotease 12) expression in these cell lines and was co-expressed in human head and neck cancers. Inhibition of HER2 or EGFR decreased ADAM12 transcripts whereas HER2 transfection upregulated ADAM12 expression. To understand the molecular mechanisms underlying HER2 regulation of ADAM12, we investigated the signaling pathways directing ADAM12 production in SCC cells. Inhibition of phosphatidyl inositol-3-kinase or mammalian target of rapamycin decreased ADAM12 transcripts in HER2-expressing SCC cells, whereas transfection with AKT increased ADAM12 mRNA. Experiments utilizing ADAM12 transfection or siRNA targeting of ADAM12 revealed that the protease increased both the migration and invasiveness of oral SCC cells. Surprisingly, ADAM12 also increased HER2 message, protein levels and activity through an Ets1-dependent mechanism. Collectively, these results reveal a novel positive activation loop between ADAM12 and HER2 that may contribute to HNSCC progression.

Previous work has shown that integrin α1-null Alport mice exhibit attenuated glomerular disease with decreased matrix accumulation and live much longer than strain-matched Alport mice. However, the mechanism underlying this observation is unknown. Here we show that glomerular gelatinase expression, specifically matrix metalloproteinase-2 (MMP-2), MMP-9, and MMP-14, was significantly elevated in both integrin α1-null mice and integrin α1-null Alport mice relative to wild-type mice; however, only MMP-9 was elevated in glomeruli of Alport mice that express integrin α1. Similarly, cultured mesangial cells from α1-null mice showed elevated expression levels of all three MMPs, whereas mesangial cells from Alport mice show elevated expression levels of only MMP-9. In both glomeruli and cultured mesangial cells isolated from integrin α1-null mice, activation of the p38 and ERK branches of the mitogen-activated protein kinase pathway was also observed. The use of small molecule inhibitors demonstrated that the activation of the p38, but not ERK, pathway was linked to elevated MMP-2, -9, and -14 expression levels in mesangial cells from integrin α1-null mice. In contrast, elevated MMP-9 levels in mesangial cells from Alport mice were linked to ERK pathway activation. Blockade of gelatinase activity using a small molecule inhibitor (BAY-12-9566) ameliorated progression of proteinuria and restored the architecture of the glomerular basement membrane in α1 integrin-null Alport mice, suggesting that elevated gelatinase activity exacerbates glomerular disease progression in these mice.

Aim: Atherosclerosis is a chronic inflammatory disease with atherosclerotic plaques containing inflammatory infiltrates. Immune cells, including T-lymphocytes, dendritic cells (DCs) and macrophages recruited from blood stream into atherosclerotic lesions, are responsible for progression and destabilization of atherosclerotic plaques. Stressed cells undergoing necrosis release molecules that act as endogenous danger signals to alert and activate innate immune cells. Upon activation, DCs, macrophages, and neutrophils produce several inflammatory cytokines and growth factors. In atherosclerotic tissue, the number of DCs increases with the progression of the lesion. In this study, we examined the expression of triggering receptor expressed on myeloid cells-1 (TREM-1) in plaque DCs and compared between the plaques from symptomatic (S) and asymptomatic (AS) patients with carotid stenosis. Methods and Results: Surgical specimens of human atherosclerotic plaques were obtained from patients undergoing carotid endarterectomy. The expression patterns of TREM-1, HLA-DR, and fascin (DC) were determined by immunofluorescence with α-actin as specific marker to identify smooth muscle cells (VSMCs). There was a marked decrease in VSMCs and an increased density of HLA-DR+ cells in the plaques of S than AS patients. Fascin expression to show DCs was undetectable in normal carotid artery specimens but significantly increased in the carotid plaques of S compared to AS. TREM-1 expression was increased in S and localized with HLA-DR and fascin. However, TREM-1 was increased in S plaques containing less number of VSMCs. The mRNA expression of MMP-9, MMP-1 was increased while Col 1 (α1) and Col III (α1) mRNA transcripts were decreased in S plaques. Conclusions: Increased expression of TREM-1 in plaque DCs of S compared to AS patients suggests a potential role of TREM-1 in atherosclerotic plaque vulnerability. Collectively, these data demonstrate that selective blockade of TREM-1 may contribute to the development of new therapies and promising targets for stabilizing atherosclerotic plaques.

Aims: Unstable atherosclerotic plaques in carotid artery are characterized by rupture of their fibrous cap, leading to transient ischemic attack or stroke. However, the underlying mechanisms are unclear. Our aim was to investigate the expression and function of triggering receptor expressed on myeloid cells-1 (TREM-1) on plaque stability involving matrix metalloproteinases (MMPs) and examine the effect of inflammatory cytokine TNF-α in symptomatic (S) compared to asymptomatic (AS) patients with carotid stenosis. Methods and Results: The mRNA transcripts for TREM-1, MMPs (MMP-1, MMP-3, and MMP-9) and collagen type I and III were analyzed by qPCR and immunofluorescence, respectively in whole plaque and plaque smooth muscle cells (pSMCs) isolated from carotid endarterectomy tissues of patients with carotid stenosis. The mRNA transcripts of TREM-1, MMP-1 and MMP-9 were increased while Col I and Col III mRNA transcripts were decreased in pSMCs of S compared to AS patients. Stimulation of pSMCs with TNF-α further increased the mRNA transcripts of TREM-1 and MMPs without any additional effect on Col I (α1) or Col III (α1) mRNA levels. The inhibitors of NF-kB (BAY11-7085), JNK (SP600125) and PI3K (LY294002) signaling pathways attenuated the effect of TNF-α. Modulation of TREM-1 in TNF-α-treated pSMCs by the administration of TREM-1 decoy receptor rTREM-1/Fc, monoclonal antibodies, or siRNA to TREM-1 attenuated the expression of MMPs and Col I (α1) and Col III (α1) in both S and AS groups. Conclusion: Increased expression of TREM-1 in pSMCs of symptomatic than in asymptomatic patients suggest a potential role of TREM-1 in plaque destabilization. This is further supported by the pronounced effect of inflammatory cytokine, TNF-α, to increase of mRNA transcripts for TREM-1 and MMPs. Selective blockade of TREM-1 may contribute to the development of new therapies and promising targets for stabilizing vulnerable atherosclerotic plaques.

We have previously demonstrated the effectiveness of adenovirus-mediated expression of antisense urokinase-type plasminogen activator receptor (uPAR) and matrix metalloproteinase-9 (MMP-9) in inhibiting tumor invasion in vitro and ex vivo. However, the therapeutic effect of the adenovirus-mediated antisense approach was shown to be transient and required potentially toxic, high viral doses. In contrast, RNA interference (RNAi)-mediated gene targeting may be superior to the traditional antisense approach, because the target mRNA is completely degraded and the molar ratio of siRNA required to degrade the target mRNA is very low. Here, we have examined the siRNA-mediated target RNA degradation of uPAR and MMP-9 in human glioma cell lines. Using RNAi directed toward uPAR and MMP-9, we achieved specific inhibition of uPAR and MMP-9. This bicistronic construct (pUM) inhibited the formation of capillary-like structures in both in vitro and in vivo models of angiogenesis. We demonstrated that blocking the expression of these genes results in significant inhibition of glioma tumor invasion in Matrigel and spheroid invasion assay models. RNAi for uPAR and MMP-9 inhibited cell proliferation, and significantly reduced the levels of phosphorylated forms of MAPK, ERK, and AKT signaling pathway molecules when compared with parental and empty vector/scrambled vector-transfected SNB19 cells. Furthermore, using RNAi to simultaneously target two proteases resulted in total regression of pre-established intracerebral tumor growth. Our results provide evidence that the use of hairpin siRNA expression vectors for uPAR and MMP-9 may provide an effective tool for cancer therapy.

Alport syndrome results from mutations in genes encoding collagen α3(IV), α4(IV), or α5(IV) and is characterized by progressive glomerular disease associated with a high-frequency sensorineural hearing loss. Earlier studies of a gene knockout mouse model for Alport syndrome noted thickening of strial capillary basement membranes in the cochlea, suggesting that the stria vascularis is the primary site of cochlear pathogenesis. Here we combine a novel cochlear microdissection technique with molecular analyses to illustrate significant quantitative alterations in strial expression of mRNAs encoding matrix metalloproteinases-2, -9, -12, and -14. Gelatin zymography of extracts from the stria vascularis confirmed these findings. Treatment of Alport mice with a small molecule inhibitor of these matrix metalloproteinases exacerbated strial capillary basement membrane thickening, demonstrating that alterations in basement membrane metabolism result in matrix accumulation in the strial capillary basement membranes. This is the first demonstration of true quantitative analysis of specific mRNAs for matrix metalloproteinases in a cochlear microcompartment. Further, these data suggest that the altered basement membrane composition in Alport stria influences the expression of genes involved in basement membrane metabolism.

Matrix metalloproteinases (MMPs) play an important regulatory role in many biological and pathological processes and their specific role in Alport syndrome (AS) is not yet clearly defined. In this study, the naturally occurring canine X-linked AS was used to demonstrate a potential role for MMP-3 and MMP-7 in Alport renal pathogenesis. Recently, we demonstrated that the expression of MMP-2, MMP-9 and MMP-14 was upregulated in the renal cortex of dogs with a spontaneous form of XLAS. In the present study, we examined necropsy samples of renal cortex from normal and XLAS dogs for MMP-3 and MMP-7 as they have the potential to activate MMP-2 and MMP-9. Immunohistochemical analysis showed strong immunostaining for both MMP-3 and MMP-7 in the interstitial space of XLAS kidneys, while virtually no immunostaining was observed in similar fields from normal dogs. RT-PCR and casein zymography confirmed that both mRNA transcripts and activities of MMP-3 and MMP-7 are elevated in XLAS kidneys. The induction of these MMPs likely contributes to tissue destruction associated with the fibrogenic process, while augmenting the activation of MMP-2 and MMP-9 by MMP-3 and MMP-7 in XLAS. Thus, these data further implicate a role for the MMPs in progressive renal pathogenesis associated with AS.

Increased expression of MMP-2, MMP-9 (type IV collagenases/gelatinases), and MT1-MMP in canine X-linked Alport syndrome (XLAS). Background Alport syndrome is a group of genetic disorders resulting from mutations in either the α3(IV), α4(IV) or α5(IV) collagen chains. The disease is characterized by a progressive glomerulonephritis, usually associated with a high-frequency specific sensorineural hearing loss, dot and fleck retinopathy, and lens abnormalities. Dogs with naturally occurring genetic disorders of basement membrane collagen (type IV) may serve as animal models of Alport syndrome. In this study, a well-characterized naturally occurring canine model was employed to demonstrate a potential role for matrix metalloproteinases (MMPs) in Alport renal disease pathogenesis. Methods Adolescent male dogs that developed renal failure were euthanized and necropsied. Clinicopathologic features of the disease were characterized, and kidneys from normal and Alport dogs were analyzed by gelatin zymography, Western blotting, in situ zymography, immunohistology, and by reverse transcription polymerase chain reaction (RT-PCR) for expression of MMP-2, MMP-9, and membrane type 1-MMP (MT1-MMP). Results Affected dogs developed proteinuria and rapidly progressive juvenile-onset chronic renal failure. The activities of MMP-2 and MMP-9 were significantly induced in Alport kidney. In situ zymography confirmed elevated active metalloproteinases in kidney cryosections of affected dogs. The mRNAs encoding MMP-2, MMP-9 and MT1-MMP were also increased in Alport dogs suggesting that elevated expression of MMPs reflects events in the progression of Alport syndrome in dogs. Conclusion Elevated expression of MMP-2, MMP-9, and MT1-MMP is observed in fibrotic renal cortex from X-linked Alport syndrome dogs. These findings suggest that MMPs may play an important role in matrix accumulation associated with progressive renal scarring in this model.

Monocytes may promote myofibroblast accumulation and apoptosis in Alport renal fibrosis. Background In interstitial fibrosis, monocytes and myofibroblasts have been directly implicated in scarring, apoptosis, and tissue necrosis. While much has been done to explore the role of these cell types individually in fibrosis, the interactive dependency of monocytes and myofibroblasts has been only marginally explored. Methods Alport mice were treated or not with a soluble receptor inhibitor for transforming growth factor-β1 (TGF-β1), which was previously shown to inhibit the accumulation of myofibroblasts, but not monocytes, in the tubulointerstitium. Kidneys were examined for fibrosis using several matrix markers, TGF-β1 mRNA expression by in situ hybridization, apoptosis using the terminal deoxynucleotidyl transferase-mediated uridine triphosphate nick end labeling (TUNEL) assay, expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPS) by dual immunofluorescence microscopy, MMP activity by gelatin and in situ zymography, MMP mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR), and basement membrane degradation by dual immunofluorescence confocal microscopy and electron microscopy. Results Treated mice showed a markedly reduced accumulation of matrix proteins. Tissue monocytes express TGF-β1 mRNA, and TGF-β1 is required for myofibroblast accumulation. The number of apoptotic cells was not influenced by TGF-β1 inhibition. Monocytes express MMP-2, MMP-9, TIMP-2, and TIMP-3. MMP activity and mRNA expression is equally up regulated in treated and untreated Alport mice. Tubular basement membranes (TBM) around clusters of monocytes are notably degraded. TGF-β1 inhibition does not extend the life of Alport mice. Conclusion These studies demonstrate that monocytes may influence myofibroblast accumulation via TGF-β1, and that monocytes, and not myofibroblasts, are associated with tubular atrophy in Alport mice. Elevated MMP expression and activity is associated with TBM destruction near monocytes clusters, suggesting an anoikis mechanism may contribute to apoptosis in this model.

Introduction: Coronary thrombosis and rupture of plaques leading to stroke are major causes of morbidity and mortality. Here, we examined the role of v-ets erythroblastosis virus E26 oncogene homologue-1 (ETS-1) on the epidermal growth factor (EGF)-induced destabilization of collagen type I (Col-Iα1) and collagen type III (Col-IIIα1) involving matrix metalloproteinase-1 (MMP-1) and MMP-9 in isolated vascular smooth muscle cells (VSMCs) from the plaques of symptomatic (S) and asymptomatic (AS) patients with carotid stenosis. To understand the molecular mechanisms underlying EGFR-regulated ETS-1, the signaling pathways directing ETS-1 production was investigated and examined if EGF-induced loss of fibrillar collagens in patients with carotid stenosis is regulated by ETS-1. Methods: VSMCs isolated from carotid plaques of both AS and S patients were treated with or without EGF. The mRNA transcripts and protein for ETS-1, MMP-9 and -1, EGFR, Col-I(α1) and Col-III(α1) were analyzed by qPCR and immunofluorescence, respectively. Results: Treatment of VSMCs with EGF significantly increased ETS-1 expression and immunoreactivity in S compared to AS. EGFR inhibitor (AG1478) decreased ETS-1 expression and upregulated Col-I(α1) and Col-III(α1) in EGF-treated VSMCs of both groups. In the transfection experiments, overexpression of ETS-1 in VSMCs increased mRNA transcripts of MMP-9 and MMP-1 and decreased the collagen mRNA transcripts. Knock-down of ETS-1 gene with either siRNA or transfection with dominant-negative form (ETS-DN) blocked EGF-induced MMP-1 and MMP-9 expression and increased mRNA transcripts for Col-I(α1) and Col-III(α1) in S compared to AS group. Inhibitors of p38MAPK (SB202190) and JNK-II (SP600125) decreased ETS-1, MMP-1 and MMP-9 transcripts and increased collagen transcripts in EGF-treated VSMCs, suggesting that p38MAPK and JNK pathways are involved in EGFR-induced collagen loss. Conclusion: These findings revealed a novel mechanism of ETS-1-regulated EGF-induced collagen loss in human carotid plaques and this could be a leading cause of plaque instability in patients with carotid stenosis. Selective blockade of ETS-1 and EGFR may be novel strategy and promising target for treating unstable and vulnerable plaques.

Solar ultraviolet (UV) radiation can cause severe damage to the skin and is the primary cause of most skin cancer. UV radiation causes DNA damage leading to mutations and also activates the Erbb2/HER2 receptor through indirect mechanisms involving reactive oxygen species. We hypothesized that Erbb2 activation accelerates the malignant progression of UV-induced skin cancer. Following the induction of benign squamous papillomas by UV exposure of v-ras(Ha) transgenic Tg.AC mice, mice were treated topically with the Erbb2 inhibitor AG825 and tumor progression monitored. AG825 treatment reduced tumor volume, increased tumor regression, and delayed the development of malignant squamous cell carcinoma (SCC). Progression to malignancy was associated with increased Erbb2 and ADAM12 (A Disintegin And Metalloproteinase 12) transcripts and protein, while inhibition of Erbb2 blocked the increase in ADAM12 message upon malignant progression. Similarly, human SCC and SCC cell lines had increased ADAM12 protein and transcripts when compared to normal controls. To determine whether Erbb2 up-regulation of ADAM12 contributed to malignant progression of skin cancer, Erbb2 expression was modulated in cultured SCC cells using forced over-expression or siRNA targeting, demonstrating up-regulation of ADAM12 by Erbb2. Furthermore, ADAM12 transfection or siRNA targeting revealed that ADAM12 increased both the migration and invasion of cutaneous SCC cells. Collectively, these results suggest Erbb2 up-regulation of ADAM12 as a novel mechanism contributing to the malignant progression of UV-induced skin cancer. Inhibition of Erbb2/HER2 reduced tumor burden, increased tumor regression, and delayed the progression of benign skin tumors to malignant SCC in UV-exposed mice. Inhibition of Erbb2 suppressed the increase in metalloproteinase ADAM12 expression in skin tumors, which in turn increased migration and tumor cell invasiveness.

The recent progress in matrix metalloproteinases (MMPs) research has opened up many cellular targets in a variety of diseases. We have discussed some of the interesting and newly described roles of MMPs in modulating metastatic phenotype in malignancies. This information suggests a more specific and exclusive role of MMPs as important regulators of tumor cell invasion and metastasis. A great deal of room exists for redefining our current understanding of the MMPs system; the more we understand about how MMPs act, the better we will understand the pathobiology of metastasis. Research in the regulation of MMPs and the potential use of MMPs for therapeutic interventions in metastasis continues to expand. The future of therapy involving inhibitors of MMPs as a part of the armamentarium against human neoplasm should be viewed with cautious optimism. This therapeutic approach is beginning to find a place in our understanding of metastatic tumors, but with perhaps few exceptions should still be considered experimental. This area of research now needs more biological knowledge and imagination on the part of investigators to make future achievements possible.

Matrix metalloproteinases (MMPs) play an important regulatory role in tissue morphogenesis, cell differentiation, tumor invasion and metastasis. Several authors have reported a direct correlation between the production of 72 kDa (MMP-2) and 92 kDa (MMP-9) type IV collagenases/gelatinases and the metastatic potential of cancer cells. Recently, we have identified the expression of both MMP-2 and MMP-9 in primary cultures of human giant cell tumor (GCT) of bone in vitro, and in tissue extracts in vivo. Interestingly, MMP-9 is not secreted by late-passaged GCT cells. It is possible that the production of MMP-9 is regulated by certain factor(s) secreted by the multinucleated giant cells in the primary culture. In order to test this hypothesis, the effect of primary-culture-conditioned medium on the expression of MMP-9 by late-passaged mononuclear stromal cells was examined. Adding conditioned medium from the primary GCT culture to the late-passaged stromal cells induced MMP-9, as evidenced by the presence of lytic bands at Mr 92000 and 72000 on a gelatin zymogram. These enzyme activities were inhibited by EDTA, a well-known inhibitor of the MMPs. We confirmed these results by Western blotting using specific antibodies and RT-PCR for MMP-2 and MMP-9. Immunofluorescence studies with specific antibodies to MMP-9 further confirmed its expression by the passaged stromal cells cultured in the primary-culture-conditioned medium. The data indicate that MMP-2 and MMP-9 are produced by the mononuclear stromal cells when cultured in GCT primary-culture-conditioned medium. This suggests that multinucleated giant cells in primary cultures secrete a factor(s) that stimulates stromal cells to produce MMP-9, which, in turn, may contribute to the aggressive behavior of GCT.

Aims Atherogenesis is a chronic progressive process that develops over several decades. Changes in the structure and composition of extracellular matrix (ECM) play an important role in the process of atherosclerosis. Matrix metalloproteinases (MMPs) and growth factors present in atherosclerotic human plaques could be related to plaque remodeling and increased risk for plaque destabilization and rupture. Hypothesis Selective inhibition of elevated MMP-9 and epidermal growth factor receptor (EGFR) increases fibrillar collagens (Type I and III) in vascular smooth muscle cells (VSMCs) from symptomatic (S) and asymptomatic (AS) patients with carotid stenosis. Methods and results Cultured human VSMCs from both AS and S patients were treated with or without epidermal growth factor (EGF). The proteolytic activity of MMP-9 and MMP-2 was quantified by gelatin zymography. The mRNA transcripts of MMP-9, MMP-1, EGFR and collagen types I (COL1A1 and COL1A2) and III (COL3A1) were analyzed by qPCR. EGF treatment significantly increased MMP-9 activity and mRNA transcripts for MMP-9, MMP-1and EGFR, while mRNA transcripts for collagen types I and III were decreased in both AS and S groups. Collagen type I and type III mRNA transcripts were significantly decreased in symptomatic VSMCs compared to cells from AS. Inhibition of EGFR with AG1478 and MMP-9 using small molecule inhibitor and transfection with MMP-9 siRNA decreased the activity and mRNA expression of MMP-9 and increased mRNA transcripts of collagen I and III in EGF treated VSMCs. Conclusion These results reveal a novel mechanism by which MMP-9 induced by EGFR activation could be a leading cause of plaque instability in patients with carotid stenosis; and the selective blockade of EGFR and MMP-9 may be a novel strategy and a promising target for treating human unstable and vulnerable plaques.

Rationale Adipose-derived mesenchymal stem cells (ADMSCs) are multipotent cells that have the potential to differentiate into different cell linages, and represent promising tools in various clinical applications. However, the molecular mechanisms that control the ability of ADMSCs to remodel 3-dimensional extracellular matrix (ECM) barriers during differentiation are not clearly understood. Herein, we studied the expression of matrix metalloproteinases (MMPs) during the differentiation of ADMSCs to endothelial cells (ECs) in vitro . Methods MSCs were isolated from porcine abdominal adipose tissue, and characterized by positive staining for MSC markers, CD44, CD73, CD90, and negative staining for CD11b, CD34 and CD45. The plasticity of MSCs was detected by bi-lineage differentiation to osteocytes, and adipocytes. The mRNA transcripts for different MMPs and TIMPs and protein expression of EC markers were analyzed by RT-PCR and immunostaining. The enzyme activity and protein expression were also analyzed by gelatin zymography, ELISA, and Western blot. Results The differentiation of ADMSCs to ECs was confirmed by the positive staining and mRNA expression of the endothelial markers. The mRNA transcripts for MMP-2 and membrane type 1 MMP (MT1-MMP) was significantly increased by 2.5 and 2.0 fold, respectively, during the differentiation of MSCs into ECs. Western blot and ELISA showed an elevated MT1-MMP and MMP-2 expression. The enzyme activity of MMP-2 was also observed by gelatin zymography. Conclusion We demonstrated that porcine ADMSCs have the ability to differentiate into ECs, and this process involves the up-regulation of MMP-2 and MT1-MMP. The increase in the expression of MMP-2 and MT1-MMP may, at least partially, facilitate the change in morphology of MSCs by degrading the ECM barriers. These findings may provide a potential mechanism for the role of MMP2 and MT1-MMP in the differentiation of ADMSCs into ECs.

Matrix metalloproteinases play an important regulatory role in tissue morphogenesis, cell differentiation and motility, and tumor cell invasiveness. We have recently demonstrated elevated activity of the 92 kDa type IV collagenase (MMP-9) in human glioblastoma and in the present study examine the relative amounts of MMP-9 protein and mRNA in human gliomas and as well as the distribution of MMP-9 in human glioma tumors in vivo. Using an enzyme-linked immunosorbent assay for the quantitative determination of MMP-9 protein, we found that levels were significantly higher in malignant astrocytomas, especially in glioblastoma multiforme, than in normal brain tissues and low-grade gliomas. In addition, the amount of MMP-9 mRNA, as determined by northern blot analysis was higher in anaplastic astrocytomas and glioblastoma multiforme than in normal brain tissue and low-grade gliomas. Immunocytochemical staining for MMP-9 showed strong cytoplasmic immunoreactivity in the tumor cells and the proliferating endothelial cells of glioblastoma multiforme and anaplastic astrocytomas. The staining intensity was lowe in low-grade astrocytomas, and was undetectable or very low in normal brain astrocytes. The results indicate that expression of MMP-9 is dramatically upregulated in highly malignant gliomas and correlates with the highly malignant progression of human gliomas in vivo, and support a role for the MMP-9 in facilitating the invasiveness seen in malignant gliomas in vivo.

Unstable carotid atherosclerotic plaques are characterized by rupture of their fibrous cap, leading to thromboembolism and stroke. The role of MMPs in plaque disruption is not well understood. The control of MMP transcription is complex but recently, EGFR expression has been implicated in the up-regulation of MMP-9. The aim of this study was to determine the MMP-9 mRNA expression as well as other MMPs (MMP-3, -7, and -14/MT1-MMP) expression in carotid plaques as well as in isolated SMCs from asymptomatic and symptomatic patients with carotid stenosis. The MMP-2 and MMP-9 activities were determined by gelatin zymography. The mRNA transcripts of MMP-9, -3, -7, -14 and EGFR were quantified by qPCR. The activity of MMP-9 on gelatin zymograms and the MMP-9 mRNA transcripts were significantly increased in symptomatic carotid plaques and isolated SMCs compared to those from plaques of asymptomatic patients. There was a parallel increase in the mRNA transcripts of MMP-7, -3 and -14. The mRNA transcripts for EGFR were also up-regulated. These results suggest that MMP-9 may be activated by MMP-7 and -3 and thus destabilizes the symptomatic plaques of patients compared to those from asymptomatic plaques and this effect may be mediated by EGFR.

Deficiency of Vitamin D is linked to an increased risk of hypertension, peripheral artery disease, and myocardial infarction and is a major risk factor for the development of human atherosclerosis. Atheromatous cytokines, including TNF-α, IL-6 and IFN-γ, and EGF receptor family growth factors are released at the site of atherosclerosis and act on proteolytic enzymes, MMPs (matrix metalloproteinases), ADAMs (a disintegrin and metalloproteinases), and ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs). ADAM-12 activates EGFR resulting in increased migration and proliferation of smooth muscle cells (SMCs). The aim of this study was to examine the effect of vitamin D on IL-6-induced ADAM-12 expression and SMC migration and proliferation. Micro-swine were fed with either vitamin D-deficient high cholesterol diet or high cholesterol diet containing 900 IU of vitamin D for 6 months. After six months when serum cholesterol levels ranged from 500-600 mg/dL, vitamin D-deficient group continued on the same deficient diet, whereas the other group received supplementation of vitamin D (1,000 IU/d) for 6 months. The mRNA expression of ADAM-12 and EGFR in whole carotid artery and in IL-6-treated SMCs was quantified by qPCR. The proliferation was assayed by CyQuant NF cell proliferation assay. The mRNA transcripts of ADAM-12 and EGFR were significantly increased in carotid arteries from Vitamin D-deficient than in vitamin D- supplemented swine. Treatment of SMCs with IL-6 also increased the mRNA transcripts of ADAM-12 and EGFR in vitamin D-deficient swine SMCs compared to vitamin D-supplemented swine SMCs. The cell proliferation was higher in SMCs isolated from Vitamin D-deficient swine carotid artery compared to vitamin D- supplemented swine carotid artery. Together, these results suggest that Vitamin D regulates ADAM-12-mediated activation of EGFR and vitamin D deficiency further enhances proliferation of SMCs, which is potentiated by atheromatous cytokines.

The question of maternal seizures as a teratogen was raised 30 years ago. Since then, a great deal of controversy has existed about the role of seizures in pregnant mothers as opposed to the direct effects of anticonvulsant medications. Animal and human research has clearly shown that anticonvulsants are teratogens and pose a risk for fetal malformations. In the case of dilantin, it appears that fetal susceptibility correlates with the fetal level of the microsomal detoxifying enzyme epoxide hydrolase.

We have examined the extent of intracellular degradation of newly synthesized collagen occurring in fibroblasts from a patient with prolidase deficiency, a rare, autosomal recessively inherited disorder, in which a lack of prolidase, which normally cleaves imidodipeptides with a C-terminal Pro or Hyp residue, results in hyperimidodipeptiduria. The main clinical feature of the condition is chronic, intractable ulceration of the skin, and the suggestion has been made that it represents a specific disorder of collagen metabolism. Although most of the hydroxy-[14]proline derived from the intracellular degradation of newly synthesized collagen in prolidase-deficient fibroblasts occurred in imidodipeptides, with a similar chromatographic profile to those occurring in the patient's urine, the proportion of collagen undergoing such degradation was as in control cells. No abnormality was found in other parameters of collagen metabolism studied, and the results confirm that, although the pathogenesis of its clinical manifestations remains unclear, the disorder is one of protein degradation in general.

The recent progress in matrix metalloproteinases (MMPs) research has opened up many cellular targets in a variety of diseases. We have discussed some of the interesting and newly described roles of MMPs in modulating metastatic phenotype in malignancies. This information suggests a more specific and exclusive role of MMPs as important regulators of tumor cell invasion and metastasis. A great deal of room exists for redefining our current understanding of the MMPs system; the more we understand about how MMPs act, the better we will understand the pathobiology of metastasis. Research in the regulation of MMPs and the potential use of MMPs for therapeutic interventions in metastasis continues to expand. The future of therapy involving inhibitors of MMPs as a part of the armamentarium against human neoplasm should be viewed with cautious optimism. This therapeutic approach is beginning to find a place in our understanding of metastatic tumors, but with perhaps few exceptions should still be considered experimental. This area of research now needs more biological knowledge and imagination on the part of investigators to make future achievements possible.

Alport glomerular disease is associated with dysregulation of pro-inflammatory cytokines and matrix metalloproteinases, promoting progressive glomerulonephritis. Changes in composition and structure of Alport GBM resulting from mutations in type IV collagen genes likely alter cell adhesion and cell signaling. Enhanced biomechanical strain on the capillary tuft, resulting from a thinner and less crosslinked GBM may be a source of insult which contributes to gene dysregulation. To test this we subjected cultured podocytes to cyclic biomechanical strain. We observed robust induction of MMP-3, −9, −10, and −14, but not MMP-2 or MMP-12. IL-6 was induced by biomechanical strain, and neutralizing antibodies against IL-6 attenuated induction of MMP-3 and MMP-10. Alport mice given L-NAME salts, which resulted in a significant rise in systolic blood pressure, showed Induction of MMP-3, MMP-10, and IL-6 in glomeruli relative to normotensive Alport mice. Hypertensive Alport mice also had elevated proteinuria, and more advanced GBM disease histologically and ultrastucturally. Collectively these data suggest MMP and cytokine dysregulation may constitute a maladaptive response to biomechanical strain in Alport podocytes, and that this response may contribute to the mechanism of glomerular disease initiation and progression.

Primary cultures of chondrocytes isolated from the proliferative and hypertrophic zones of bovine growth plate cartilage were established, and their morphological and biosynthetic characteristics were com- pared. High-cell-density monolayer cultures maintained a predominantly chondrocytic morphology, preserved their zone-specific collagen phenotype, and produced an abundant proteoglycan matrix. In contrast, monolayer cultures plated at low cell densities contained many cells with fibroblastic morphology and initiated the synthesis of type I collagen within 72 h following seeding. On a per cell basis, the proliferative zone chondrocytes displayed greater biosynthetic activity than chondrocytes from the hypertrophic region. Characterization of newly synthesized collagens showed that type II collagen was the major biosynthetic product of both types of cells. In contrast, type X collagen was synthesized exclusively by hypertrophic chondrocytes and represented about 20% of the total collagen produced by these cells. The type X collagen molecules synthesized by the hypertrophic cells contained disulfide bonds within their triple-helical domains and assembled into a high-Mr aggregate. When polymerase chain reaction (PCR) was employed to amplify type X collagen sequences from cDNA obtained by reverse transcription of mRNA from cul- tures of both types of chondrocytes, only the hypertrophic chondrocyte mRNA yielded PCR amplification products indicative of the presence of type X collagen transcripts.

Previous work has shown that integrin alpha1-null Alport mice exhibit attenuated glomerular disease with decreased matrix accumulation and live much longer than strain-matched Alport mice. However, the mechanism underlying this observation is unknown. Here we show that glomerular gelatinase expression, specifically matrix metalloproteinase-2 (MMP-2), MMP-9, and MMP-14, was significantly elevated in both integrin alpha1-null mice and integrin alpha1-null Alport mice relative to wild-type mice; however, only MMP-9 was elevated in glomeruli of Alport mice that express integrin alpha1. Similarly, cultured mesangial cells from alpha1-null mice showed elevated expression levels of all three MMPs, whereas mesangial cells from Alport mice show elevated expression levels of only MMP-9. In both glomeruli and cultured mesangial cells isolated from integrin alpha1-null mice, activation of the p38 and ERK branches of the mitogen-activated protein kinase pathway was also observed. The use of small molecule inhibitors demonstrated that the activation of the p38, but not ERK, pathway was linked to elevated MMP-2, -9, and -14 expression levels in mesangial cells from integrin alpha1-null mice. In contrast, elevated MMP-9 levels in mesangial cells from Alport mice were linked to ERK pathway activation. Blockade of gelatinase activity using a small molecule inhibitor (BAY-12-9566) ameliorated progression of proteinuria and restored the architecture of the glomerular basement membrane in alpha1 integrin-null Alport mice, suggesting that elevated gelatinase activity exacerbates glomerular disease progression in these mice.

The cause of the Ehlers-Danlos syndrome Type VII (EDS VII) is considered to be defective removal of the amino-terminal propeptide (N-propeptide) of Type I procollagen due to deficiency of procollagen N-proteinase, the enzyme responsible for the normal proteolytic excision of this precursor-specific domain. Molecules retaining the N-propeptide (pN-collagen molecules) are thought to cause defective fibrillogenesis and cross-linking which eventuate in dramatic joint laxity and joint dislocations, the clinical hallmark of this variety of EDS. Recent studies demonstrate that some EDS VII patients harbor small deletions of either the pro-alpha 1(I) or pro-alpha 2(I) chain of Type I procollagen. We have found an 18-amino acid deletion (due to exon outsplicing) in a mutant pro-alpha 2(I) chain from such a patient. The deleted peptide is the junctional segment (N-telopeptide) linking the alpha 2(I) N-propeptide and major triple helical domains; loss of this short segment results in union of these latter domains and produces a shortened pN alpha 2(I) chain. Directly extracted tissue collagen and pepsin-digested fibroblast collagen contain this mutant pN alpha 2(I) chain and normal alpha 1(I) chains, but not pN alpha 1(I) chains, indicating that the relatively larger alpha 1(I) N-propeptide is excised from the related alpha 1(I) chains. The fate of this alpha 1(I) N-propeptide was unclear and therefore whether or not the intact N-propeptide was, in fact, retained in native mutant collagen was also unclear. In this paper, we describe morphologic, chemical, and immunochemical studies which indicate that the alpha 1(I) N-propeptide is retained in noncovalent association with the mutant pN alpha 2(I) chain in native mutant collagen molecules both in vivo and in vitro. In both instances, the alpha 1(I) N-propeptides are proteolytically cleaved from the related alpha 1(I) chains. These data suggest that retention of a partially cleaved, but essentially intact N-propeptide in mutant collagen may play a role in the pathogenesis of this disease.

Alport syndrome is a glomerular basement membrane (GBM) disease caused by mutations in type IV collagen genes. A unique irregular thickening and thinning of the GBM characterizes the progressive glomerular pathology. The metabolic imbalances responsible for these GBM irregularities are not known. Here we show that macrophage metalloelastase (MMP-12) expression is >40-fold induced in glomeruli from Alport mice and is markedly induced in glomeruli of both humans and dogs with Alport syndrome. Treatment of Alport mice with MMI270 (CGS27023A), a broad spectrum MMP inhibitor that blocks MMP-12 activity, results in largely restored GBM ultrastructure and function. Treatment with BAY-129566, a broad spectrum MMP inhibitor that does not inhibit MMP-12, had no effect. We show that inhibition of CC chemokine receptor 2 (CCR2) receptor signaling with propagermanium blocks induction of MMP-12 mRNA and prevents GBM damage. CCR2 receptor is expressed in glomerular podocytes of Alport mice, suggesting MCP-1 activation of CCR2 on podocytes may underlie induction of MMP-12. These data indicate that the irregular GBM that characterizes Alport syndrome may be mediated, in part, by focal degradation of the GBM due to MMP dysregulation, in particular, MMP-12. Thus, MMP-12/CCR2 inhibitors may provide a novel and effective therapeutic stra-tegy for Alport glomerular disease.

Background Glomerular basement membrane (GBM), a key component of the blood-filtration apparatus in the in the kidney, is formed through assembly of type IV collagen with laminins, nidogen, and sulfated proteoglycans. Mutations or deletions involving α3(IV), α4(IV), or α5(IV) chains of type IV collagen in the GBM have been identified as the cause for Alport syndrome in humans, a progressive hereditary kidney disease associated with deafness. The pathological mechanisms by which such mutations lead to eventual kidney failure are not completely understood. Methods and Findings We showed that increased susceptibility of defective human Alport GBM to proteolytic degradation is mediated by three different matrix metalloproteinases (MMPs)—MMP-2, MMP-3, and MMP-9—which influence the progression of renal dysfunction in α3(IV) −/− mice, a model for human Alport syndrome. Genetic ablation of either MMP-2 or MMP-9, or both MMP-2 and MMP-9, led to compensatory up-regulation of other MMPs in the kidney glomerulus. Pharmacological ablation of enzymatic activity associated with multiple GBM-degrading MMPs, before the onset of proteinuria or GBM structural defects in the α3(IV) −/− mice, led to significant attenuation in disease progression associated with delayed proteinuria and marked extension in survival. In contrast, inhibition of MMPs after induction of proteinuria led to acceleration of disease associated with extensive interstitial fibrosis and early death of α3(IV) −/− mice. Conclusions These results suggest that preserving GBM/extracellular matrix integrity before the onset of proteinuria leads to significant disease protection, but if this window of opportunity is lost, MMP-inhibition at the later stages of Alport disease leads to accelerated glomerular and interstitial fibrosis. Our findings identify a crucial dual role for MMPs in the progression of Alport disease in α3(IV) −/− mice, with an early pathogenic function and a later protective action. Hence, we propose possible use of MMP-inhibitors as disease-preventive drugs for patients with Alport syndrome with identified genetic defects, before the onset of proteinuria., In a mouse model of Alport disease, matrix metalloproteinases appear to have a crucial dual role in disease progression, with an early pathogenic function and a later protective action.

Alport syndrome results from mutations in genes encoding collagen alpha3(IV), alpha4(IV), or alpha5(IV) and is characterized by progressive glomerular disease associated with a high-frequency sensorineural hearing loss. Earlier studies of a gene knockout mouse model for Alport syndrome noted thickening of strial capillary basement membranes in the cochlea, suggesting that the stria vascularis is the primary site of cochlear pathogenesis. Here we combine a novel cochlear microdissection technique with molecular analyses to illustrate significant quantitative alterations in strial expression of mRNAs encoding matrix metalloproteinases-2, -9, -12, and -14. Gelatin zymography of extracts from the stria vascularis confirmed these findings. Treatment of Alport mice with a small molecule inhibitor of these matrix metalloproteinases exacerbated strial capillary basement membrane thickening, demonstrating that alterations in basement membrane metabolism result in matrix accumulation in the strial capillary basement membranes. This is the first demonstration of true quantitative analysis of specific mRNAs for matrix metalloproteinases in a cochlear microcompartment. Further, these data suggest that the altered basement membrane composition in Alport stria influences the expression of genes involved in basement membrane metabolism.

Mechanisms underlying the rupture of atherosclerotic plaque, a crucial factor in the development of myocardial infarction and stroke, are not well defined. Here, we examined the role of epidermal growth factor (EGF)‐mediated matrix metalloproteinases (MMP) on the stability of interstitial collagens in vascular smooth muscle cells (VSMCs) isolated from carotid endarterectomy tissues of symptomatic and asymptomatic patients with carotid stenosis. VSMCs isolated from the carotid plaques of both asymptomatic and symptomatic patients were treated with EGF. The MMP‐9 activity was quantified by gelatin zymography and the analysis of mRNA transcripts and protein for MMP‐9, MMP‐1, EGFR and collagen types I, Col I(α1) and collagen type III, Col III(α1) were analyzed by qPCR and immunofluorescence, respectively. The effect of EGF treatment to increase MMP‐9 activity and mRNA transcripts for MMP‐9, MMP‐1, and EGFR and to decrease mRNA transcripts for Col I(α1) and Col III(α1) was threefold to fourfold greater in VSMCs isolated from the carotid plaques of symptomatic than asymptomatic patients. Inhibitors of EGFR (AG1478) and a small molecule inhibitor of MMP‐9 decreased the MMP9 expression and upregulated Col I(α1) and Col III(α1) in EGF‐treated VSMCs of both groups. Additionally, the magnitude in decreased MMP‐9 mRNA and increased Col I(α1) and Col III(α1) due to knockdown of MMP‐9 gene with siRNA in EGF‐treated VSMCs was significantly greater in the symptomatic group than the asymptomatic group. Thus, a selective blockade of both EGFR and MMP‐9 may be a novel strategy and a promising target for stabilizing vulnerable plaques in patients with carotid stenosis., This report described the underlying mechanisms by which MMP‐1 and MMP‐9 induced by EFGR activation decreases the interstitial collagens and this could result in plaque instability in patients with carotid stenosis. Thus, selective blockade of EGFR and/or MMP‐9 may be a novel strategy and a promising target to stabilize atherosclerotic plaques and thus decreases morbidity and mortality.

Giant cell tumor (GCT) of bone is a progressive, potentially malignant process that destroys skeletal tissue. It consists of multinucleated giant cells, which are hypothesized to be derived from a monocyte/macrophage lineage and mononuclear stromal cells, and the precise relationship of these cells is not fully understood. Recently, we demonstrated that the production of matrix metalloproteinase-9 (MMP-9) in GCT stromal cells is regulated by certain factor(s) secreted by the multinucleated giant cells. In the present study, we evaluated for the presence of interleukin-1beta (IL-1beta) and attempted to establish its possible role for the induction of MMP-9 in GCT stromal cells. Using enzyme-linked immunosorbent assay (ELISA), we have demonstrated that the primary GCT cultures secrete both IL-1beta and MMP-9. The addition of monoclonal antibody (mAb) against IL-1beta partially abrogated, but did not abolish, MMP-9 expression. Our results on gelatin zymography, reverse transcriptase-polymerase chain reaction (RT-PCR), and immunofluorescence showed that GCT stromal cells did not express MMP-9, although treatment with IL-1beta induced MMP-9 expression in a dose-dependent manner, and the secretion peaked 24 h after stimulation and then plateaued. Studies with cycloheximide, a protein synthesis inhibitor, demonstrated that de novo protein synthesis is required for IL-1beta induced MMP-9 expression. Moreover, nuclear run-on analysis has revealed that IL-1beta significantly increased MMP-9 gene transcription in GCT stromal cells. The data suggest that IL-1beta secreted by the multinucleated giant cells in GCT may be one of the factors responsible for the induction of MMP-9 at the transcriptional level in GCT stromal cells in vivo. We conclude that GCT has a self-stimulatory system for the production of MMP-9, and the ability of stromal cells to produce MMP-9 with appropriate stimuli, such as IL-1beta, and possibly in concert with other cytokines may contribute to the aggressive and potentially malignant behavior of GCT.

We determined whether certain factor(s) secreted by multinucleated giant cells, which is of monocyte/macrophage lineage in giant cell tumor of bone (GCT), regulate the induction of matrix metalloproteinase (MMP)-9 expression in mononucleated stromal cells. Our data derived using enzyme linked immunosorbant assays (ELISAs) suggest that the GCT cells in primary culture produce both MMP-9 and tumor necrosis factor-alpha (TNF-alpha). Further, the MMP-9 expression in GCT primary cultures was partially abrogated by neutralizing antibody to TNF-alpha, suggesting that TNF-alpha secretion by the multinucleated giant cells may be one of the factors responsible for the production of MMP-9 by the stromal cells in vivo. In order to confirm this we examined the role of TNF-alpha on the induction of MMP-9 expression in bone GCT stromal cells. These cells express MMP-2, but not MMP-9. However, treatment of these cells with TNF-alpha induced the expression of MMP-9 in a concentration-dependent manner. Kinetic experiments revealed that the secretion of MMP-9 peaked 12 h post TNF-alpha stimulation. Immunofluorescence studies confirmed the expression of MMP-9 after stimulation of GCT stromal cells with TNF-alpha. Further, TNF-alpha-induced MMP-9 expression was completely blocked with neutralizing antibody to TNF-alpha, thereby demonstrating the specificity. In addition, the induction of MMP-9 expression by TNF-alpha was completely abrogated in the presence of cycloheximide, a protein synthesis inhibitor, suggesting that de novo protein synthesis may be required. Nuclear run-on analysis demonstrated that treatment of GCT stromal cells significantly enhanced the MMP-9 gene transcription. Together, our data suggest that TNF-alpha secreted by the multinucleated giant cells up-regulates MMP-9 expression in GCT stromal cells by the induction of certain transcription factors, which in turn enhanced the rate of transcription of MMP-9 gene. These studies also suggest the existence of an essential cell-cell interaction in the regulation of MMP-9 expression in GCT.

Objectives: To investigate whether the advanced bone age in Sotos syndrome is associated with alterations in type I collagen metabolism in bone. Design and methods: The metabolism of collagen was studied by analyzing the production, gene expression and degradation of type I collagen in dermal fibroblast strains from patients with Sotos syndrome and comparing them with fibroblasts from age-matched healthy subjects. Collagen production was determined as collagenase digestible radioactivity and collagen mRNA levels were measured by RT-PCR. Collagen degradation was assessed by specific collagenase assay and gelatin zymography. To determine the structural defects in type I collagen, the newly synthesized proteins were analyzed by SDS-PAGE before and after proteolytic digestion with pepsin. Results: In the present study, we have demonstrated that the collagen production, secretion and degradation in Sotos syndrome is comparable to controls. In addition, no qualitative differences in mRNA transcripts for type I collagen were detected between the control and Sotos syndrome fibroblasts. The secretion and intracellular accumulation of procollagen is also comparable to controls. The analysis of both procollagen and collagen on SDS-PAGE did not exhibit any major structural changes as compared with controls. Conclusions: Our results on several aspects of collagen metabolism have demonstrated for the first time that collagen, the most abundant of mammalian proteins and the major constituent of bone, is normal in patients with Sotos syndrome. Therefore, it appears that the advanced bone age and accelerated linear growth seen in patients with Sotos syndrome may not be attributed to inherent abnormalities of collagen metabolism. The etiology and the pathogenesis of Sotos syndrome still remains unclear.

The plasminogen activation system consists of plasminogen activators and their inhibitors, serine proteases, and serpins. The proteases and inhibitors regulate a variety of processes in tissue morphogenesis, differentiation, cell migration, and cancer cell invasiveness and metastasis. One of the plasminogen activators, urokinase-type plasminogen activator (uPA), binds to a specific surface and provides a localized cell surface proteolytic activity required for the destruction of extracellular matrix, which is a vital step in tumor cell invasion. The proteolytic activity of uPA is modulated by its cell surface receptor, as well as by plasminogen activator inhibitor type-1 (PAI-1) and, to a lesser degree, by other inhibitors. The role of plasminogen activators and their inhibitors in cancer invasion can be demonstrated in the development and progression of malignant brain tumors. Our findings indicate that uPA and PAI-1 expression are dramatically upregulated in malignant brain tumors in parallel with the histological progression of the tumors. The results suggest that these molecules may contribute to tumor invasion in addition to their significant role in angiogenesis. An evaluation of the plasminogen activation system could add diagnostic and prognostic significance to the evaluation of individual patients.

The molecular basis for Osteogenesis Imperfecta in a large kindred with a highly variable phenotype was identified by sequencing the mutant pro alpha 1 (I) protein, cDNA and genomic DNA from the proband. Fibroblasts from different affected individuals all synthesize both normal Type I procollagen molecules and abnormal Type I procollagen molecules in which one or both pro alpha 1 (I) chain(s) contain a cysteine residue within the triple helical domain. Protein studies of the proband localized the mutant cysteine residue to the alpha 1 (I) CB 8 peptide. We now report that cysteine has replaced glycine at triple helical residue 175 disrupting the invariant Gly-X-Y structural motif required for perfect triple helix formation. The consequences include post-translational overmodification, decreased thermal stability, and delayed secretion of mutant molecules. The highly variable phenotype in the present kindred cannot be explained solely on the basis of the cysteine for glycine substitution but will require further exploration.

A patient with Ehlers-Danlos syndrome Type VIIB was found to have an interstitial deletion of 18 amino acids in approximately half of the pro-alpha 2(I) chains of Type I procollagen. Analysis of pepsin-solubilized tissue and fibroblast collagen revealed an abnormal additional chain, alpha 2(I)', which migrated in sodium dodecyl sulfate-5% polyacrylamide gel electrophoresis between the normal alpha 1(I) and alpha 2(I) chains. The apparent ratio of normal alpha 1(I):mutant alpha 2(I)':normal alpha 2(I) was 4:1:1. Procollagen studies and enzyme digestion studies of native mutant collagen suggested defective removal of the amino propeptide. Sieve chromatography of CNBr peptides from purified alpha 2(I)' chains revealed the absence of the normal amino telopeptide fragment CB 1 and the appearance of a larger new peptide of approximately 60 residues (CB X). Compositional and sequencing studies of this peptide identified normal amino propeptide sequences. However, the most carboxyl-terminal tryptic peptide of CB X differed substantially in composition and sequence from the expected and was found to have an interstitial deletion of 18 amino acids corresponding to the N-telopeptide of the pro-alpha 2(I) chain. This deletion removes the normal sites of cleavage of the N-proteinase and also removes a critical cross-linking lysine residue. The 18 amino acids deleted correspond exactly to the residues encoded by exon 6 of the pro-alpha 2(I) collagen gene (COL 1 A2), and, therefore, the protein defect may be due to a genomic deletion, or alternatively, an RNA splicing defect.

Fibroblasts from many patients with osteogenesis imperfecta (OI) synthesize and secrete Type I collagen which is both overmodified and exhibits a decreased thermal denaturation temperature. We have examined the relationship between overmodification and decreased melting temperature in several favorable OI mutants by selectively inhibiting lysyl hydroxylase activity with the drug Minoxidil and comparing the melting profiles of the resultant undermodified collagen with untreated control. Minoxidil treatment causes an appreciable decrease in hydroxylysine with compensatory increases in lysine content, and the delayed sodium dodecyl sulfate-polyacrylamide gel electrophoretic mobility of the overmodified collagen chains becomes normal. However, the decreased melting temperature was unchanged from untreated OI control. When unhydroxylated collagen produced by normal control and OI fibroblasts incubated with alpha,alpha'-dipyridyl was examined, mutant OI molecules melted at a lower temperature than control. These data indicate that the decreased thermal denaturation temperature of OI mutant collagen is independent of post-translational overmodification of lysine or hydroxylysine. Presumably, substitutions for glycine in the Gly-X-Y structural motif distort the helix and produce lower melting temperatures by presently unknown mechanisms.

Two siblings born to consanguineous parents are reported with typical clinical features of the Ehlers-Danlos syndrome type IV. However, their cultured skin fibroblasts synthesize and secrete procollagen type III in normal amounts and proportions. This is probably a new form of the Ehlers-Danlos syndrome with autosomal recessive inheritance classified as Ehlers-Danlos syndrome type IV D.