The Nonproteolytic Intracellular Domain of Membrane-Type 1 Matrix Metalloproteinase Coordinately Modulates Abdominal Aortic Aneurysm and Atherosclerosis in Mice-Brief Report.

BACKGROUND: MT1-MMP (membrane-type 1 matrix metalloproteinase, MMP-14) is a transmembrane-anchored protein with an extracellular proteinase domain and a cytoplasmic tail devoid of proteolytic functions but capable of mediating intracellular signaling that regulates tissue homeostasis. MT1-MMP extracellular proteolytic activity has been shown to regulate pathological remodeling in aortic aneurysm and atherosclerosis. However, the role of the nonproteolytic intracellular domain of MT1-MMP in vascular remodeling in abdominal aortic aneurysms (AAA) is unknown. METHODS: We generated a mutant mouse that harbors a point mutation (Y573D) in the MT1-MMP cytoplasmic domain that abrogates the MT1-MMP signaling function without affecting its proteolytic activity. These mice and their control wild-type littermates were subjected to experimental AAA modeled by angiotensin II infusion combined with PCSK9 (proprotein convertase subtilisin/kexin type 9) overexpression and high-cholesterol feeding. RESULTS: The mutant mice developed more severe AAA than the control mice, with concomitant generation of intraaneurysmal atherosclerotic lesions and dramatically increased macrophage infiltration and elastin degradation. Aortic lesion-associated and bone marrow-derived macrophages from the mutant mice exhibited an enhanced inflammatory state and expressed elevated levels of proinflammatory Netrin-1, a protein previously demonstrated to promote both atherosclerosis and AAA. CONCLUSIONS: Our findings show that the cytoplasmic domain of MT1-MMP safeguards from AAA and atherosclerotic plaque development through a proteolysis-independent signaling mechanism associated with Netrin-1 expression. This unexpected function of MT1-MMP unveils a novel mechanism of synchronous onset of AAA and atherogenesis and highlights its importance in the control of vascular wall homeostasis.

Membrane-type 1 Matrix Metalloproteinase Modulates Tissue Homeostasis by a Non-proteolytic Mechanism.

Membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14), a transmembrane proteinase with a short cytoplasmic tail, is a major effector of extracellular matrix remodeling. Genetic silencing of MT1-MMP in mouse (Mmp14 -/- ) and man causes dwarfism, osteopenia, arthritis, and lipodystrophy, abnormalities ascribed to defective collagen turnover. We have previously shown non-proteolytic functions of MT1-MMP mediated by its cytoplasmic tail, where the unique tyrosine (Y573) controls intracellular signaling. The Y573D mutation blocks TIMP-2/MT1-MMP-induced Erk1/2 and Akt signaling without affecting proteolytic activity. Here, we report that a mouse with the MT1-MMP Y573D mutation (Mmp14 Y573D/Y573D ) shows abnormalities similar to but also different from those of Mmp14 -/- mice. Skeletal stem cells (SSC) of Mmp14 Y573D/Y573D mice show defective differentiation consistent with the mouse phenotype, which is rescued by wild-type SSC transplant. These results provide the first in vivo demonstration that MT1-MMP modulates bone, cartilage, and fat homeostasis by controlling SSC differentiation through a mechanism independent of proteolysis.

Periostin interaction with discoidin domain receptor-1 (DDR1) promotes cartilage degeneration.

Osteoarthritis (OA) is characterized by progressive loss of articular cartilage accompanied by the new bone formation and, often, a synovial proliferation that culminates in pain, loss of joint function, and disability. However, the cellular and molecular mechanisms of OA progression and the relative contributions of cartilage, bone, and synovium remain unclear. We recently found that the extracellular matrix (ECM) protein periostin (Postn, or osteoblast-specific factor, OSF-2) is expressed at high levels in human OA cartilage. Multiple groups have also reported elevated expression of Postn in several rodent models of OA. We have previously reported that in vitro Postn promotes collagen and proteoglycan degradation in human chondrocytes through AKT/ß-catenin signaling and downstream activation of MMP-13 and ADAMTS4 expression. Here we show that Postn induces collagen and proteoglycan degradation in cartilage by signaling through discoidin domain receptor-1 (DDR1), a receptor tyrosine kinase. The genetic deficiency or pharmacological inhibition of DDR1 in mouse chondrocytes blocks Postn-induced MMP-13 expression. These data show that Postn is signaling though DDR1 is mechanistically involved in OA pathophysiology. Specific inhibitors of DDR1 may provide therapeutic opportunities to treat OA.

Matrix Metalloproteinase Inhibitors in Cancer Therapy: Turning Past Failures Into Future Successes.

The matrix metalloproteinases (MMP) are a family of proteolytic enzymes that degrade multiple components of the extracellular matrix. A large body of experimental and clinical evidence has implicated MMPs in tumor invasion, neoangiogenesis, and metastasis, and therefore they represent ideal pharmacologic targets for cancer therapy. From the 1990s to early 2000s, synthetic inhibitors of MMPs (MMPI) were studied in various cancer types. Unexpectedly, despite strongly promising preclinical data, all trials were unsuccessful in reducing tumor burden or improving overall survival; in addition, MMPIs had unforeseen, severe side effects. Two main reasons can explain the failure of MMPIs in clinical trials. It has now become apparent that some MMPs have antitumor effects; therefore, the broad-spectrum MMPIs used in the initial trials might block these MMPs and result in tumor progression. In addition, although MMPs are involved in the early stages of tumor progression, MMPIs were tested in patients with advanced disease, beyond the stage when these compounds could be effective. As more specific MMPIs are now available, MMP targeting could be reconsidered for cancer therapy; however, new trials should be designed to test their antimetastatic properties in early-stage tumors, and endpoints should focus on parameters other than decreasing metastatic tumor burden. Mol Cancer Ther; 17(6); 1147-55. ©2018 AACR.

Arrested Development: Infantile Hemangioma and the Stem Cell Teratogenic Hypothesis.

BACKGROUND: Early-life programming is defined by the adaptive changes made by the fetus in response to an adverse in utero environment. Infantile hemangioma (IH), a vascular anomaly, is the most common tumor of infancy. Here we take IH as the tumor model to propose the stem cell teratogenic hypothesis of tumorigenesis and the potential involvement of the immune system. OBJECTIVES: Teratogenic agents include chemicals, heavy metals, pathogens, and ionizing radiation. To investigate the etiology and pathogenesis of IH, we hypothesized that they result from a teratogenic mechanism. Immature, incompletely differentiated, dysregulated progenitor cells (multipotential stem cells) are arrested in development with vasculogenic, angiogenic, and tumorigenic potential due to exposure to teratogenic agents such as extrinsic factors that disrupt intrinsic factors via molecular mimicry. During the critical period of immunological tolerance, environmental exposure to immunotoxic agents may harness the teratogenic potential in the developing embryo or fetus and modify the early-life programming algorithm by altering normal fetal development, causing malformations, and inducing tumorigenesis. Specifically, exposure to environmental agents may interfere with physiological signaling pathways and contribute to the generation of IH, by several mechanisms. DISCUSSION: An adverse in utero environment no longer serves as a sustainable environment for proper embryogenesis and normal development. Targeted disruption of stem cells by extrinsic factors can alter the genetic program. CONCLUSIONS: This article offers new perspectives to stimulate discussion, explore novel experimental approaches (such as immunotoxicity/vasculotoxicity assays and novel isogenic models), and to address the questions raised to convert the hypotheses into nontoxic, noninvasive treatments.

Infantile Hemangioma Originates From A Dysregulated But Not Fully Transformed Multipotent Stem Cell.

Infantile hemangioma (IH) is the most common tumor of infancy. Its cellular origin and biological signals for uncontrolled growth are poorly understood, and specific pharmacological treatment is unavailable. To understand the process of hemangioma-genesis we characterized the progenitor hemangioma-derived stem cell (HemSC) and its lineage and non-lineage derivatives. For this purpose we performed a high-throughput (HT) phenotypic and gene expression analysis of HemSCs, and analyzed HemSC-derived tumorspheres. We found that IH is characterized by high expression of genes involved in vasculogenesis, angiogenesis, tumorigenesis and associated signaling pathways. These results show that IH derives from a dysregulated stem cell that remains in an immature, arrested stage of development. The potential biomarkers we identified can afford the development of diagnostic tools and precision-medicine therapies to "rewire" or redirect cellular transitions at an early stage, such as signaling pathways or immune response modifiers.

Inhibition of Breast Cancer Metastasis by Presurgical Treatment with an Oral Matrix Metalloproteinase Inhibitor: A Preclinical Proof-of-Principle Study.

Breast cancer has the second highest death toll in women worldwide, despite significant progress in early diagnosis and treatments. The main cause of death is metastatic disease. Matrix metalloproteinases (MMP) are required for the initial steps of metastasis, and have therefore been considered as ideal pharmacologic targets for antimetastatic therapy. However, clinical trials of MMP inhibitors were unsuccessful. These trials were conducted in patients with advanced disease, beyond the stage when these compounds could have been effective. We hypothesized that early treatment with a selective MMP inhibitor between the time of diagnosis and definitive surgery, the so-called "window-of-opportunity," can inhibit metastasis and thereby improve survival. To investigate our hypothesis, we used the 4T1 mouse model of aggressive mammary carcinoma. We treated the animals with SD-7300, an oral inhibitor of MMP-2, -9, and -13, starting after the initial detection of the primary tumor. Seven days later, the primary tumors were excised and analyzed for MMP activity, and the SD-7300 treatment was discontinued. After 4 weeks, the animals were sacrificed and their lungs analyzed histologically for number of metastases and metastatic burden (metastases' area/lung section area). SD-7300 treatment inhibited 70% to 80% of tumor-associated MMP activity (P = 0.0003), reduced metastasis number and metastatic burden by 50% to 60% (P = 0.002 and P = 0.0082, respectively), and increased survival (92% vs. 66.7%; P = 0.0409), relative to control vehicle. These results show that treatment of early invasive breast cancer with selective MMP inhibitors can lower the risk of recurrence and increase long-term disease-free survival. Mol Cancer Ther; 15(10); 2370-7. ©2016 AACR.

Mitral valve prolapse is associated with altered extracellular matrix gene expression patterns.

Mitral valve prolapse (MVP) is the leading indication for isolated mitral valve surgery in the United States. Disorganization of collagens and glycosaminoglycans in the valvular extracellular matrix (ECM) are histological hallmarks of MVP. We performed a transcriptome analysis to study the alterations in ECM-related gene expression in humans with sporadic MVP. Mitral valve specimens were obtained from individuals undergoing valve repair for MVP (n=7 patients) and from non-beating heart-tissue donors (n=3 controls). Purified RNA was subjected to whole-transcriptome microarray analysis. Microarray results were validated by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Gene ontology enrichment analysis was performed. 2046 unique genes showed significant differential expression (false discovery rate <0.5%). After demonstrating appropriate sample clustering, microarray results were globally validated using a subset of 22 differentially expressed genes by RT-qPCR (Pearson's correlation r=0.65, p=0.001). Gene ontology enrichment analyses performed with ErmineJ and DAVID Bioinformatics Database demonstrated overrepresentation of ECM components (p<0.05). Functional annotation clustering calculated enrichment of ECM-related ontology groups (enrichment score=4.1). ECM-related gene expression is significantly altered in MVP. Our study is consistent with the histologically observed alterations in collagen and mucopolysaccharide profiles of myxomatous mitral valves. Furthermore, whole-transcriptome analyses suggest dysregulation of multiple pathways, including TGF-beta signaling.

Urokinase Receptor Promotes Skin Tumor Formation by Preventing Epithelial Cell Activation of Notch1.

The urokinase-type plasminogen activator receptor (uPAR) has a well-established role in cancer progression, but it has been little studied at earlier stages of cancer initiation. Here, we show that uPAR deficiency in the mouse dramatically reduces susceptibility to the classical two-stage protocol of inflammatory skin carcinogenesis. uPAR genetic deficiency decreased papilloma formation and accelerated keratinocyte differentiation, effects mediated by Notch1 hyperactivation. Notably, Notch1 inhibition in uPAR-deficient mice rescued their susceptibility to skin carcinogenesis. Clinically, we found that human differentiated keratoacanthomas expressed low levels of uPAR and high levels of activated Notch1, with opposite effects in proliferating tumors, confirming the relevance of the observations in mice. Furthermore, we found that TACE-dependent activation of Notch1 in basal kerantinocytes was modulated by uPAR. Mechanistically, uPAR sequestered TACE within lipid rafts to prevent Notch1 activation, thereby promoting cell proliferation and tumor formation. Given that uPAR signaling is nonessential for normal epidermal homeostasis, our results argue that uPAR may present a promising disease-specific target for preventing skin cancer development.

TIMP-2 Interaction with MT1-MMP Activates the AKT Pathway and Protects Tumor Cells from Apoptosis.

Membrane-type 1 matrix metalloproteinase (MT1-MMP), a transmembrane proteinase with an extracellular catalytic domain and a short cytoplasmic tail, degrades a variety of extracellular matrix (ECM) components. In addition, MT1-MMP activates intracellular signaling through proteolysis-dependent and independent mechanisms. We have previously shown that binding of tissue inhibitor of metalloproteinases-2 (TIMP-2) to MT1-MMP controls cell proliferation and migration, as well as tumor growth in vivo by activating the Ras-extracellular signal regulated kinase-1 and -2 (ERK1/2) pathway through a mechanism that requires the cytoplasmic but not the proteolytic domain of MT1-MMP. Here we show that in MT1-MMP expressing cells TIMP-2 also induces rapid and sustained activation of AKT in a dose- and time-dependent manner and by a mechanism independent of the proteolytic activity of MT1-MMP. Fibroblast growth factor receptor-1 mediates TIMP-2 induction of ERK1/2 but not of AKT activation; however, Ras activation is necessary to transduce the TIMP-2-activated signal to both the ERK1/2 and AKT pathways. ERK1/2 and AKT activation by TIMP-2 binding to MT1-MMP protects tumor cells from apoptosis induced by serum starvation. Conversely, TIMP-2 upregulates apoptosis induced by three-dimensional type I collagen in epithelial cancer cells. Thus, TIMP-2 interaction with MT1-MMP provides tumor cells with either pro- or anti-apoptotic signaling depending on the extracellular environment and apoptotic stimulus.

Elevated expression of periostin in human osteoarthritic cartilage and its potential role in matrix degradation via matrix metalloproteinase-13.

We investigated the role of periostin, an extracellular matrix protein, in the pathophysiology of osteoarthritis (OA). In OA, dysregulated gene expression and phenotypic changes in articular chondrocytes culminate in progressive loss of cartilage from the joint surface. The molecular mechanisms underlying this process are poorly understood. We examined periostin expression by immunohistochemical analysis of lesional and nonlesional cartilage from human and rodent OA knee cartilage. In addition, we used small interfering (si)RNA and adenovirus transduction of chondrocytes to knock down and up-regulate periostin levels, respectively, and analyzed its effect on matrix metalloproteinase (MMP)-13, a disintegrin and MMP with thrombospondin motifs (ADAMTS)-4, and type II collagen expression. We found high periostin levels in human and rodent OA cartilage. Periostin increased MMP-13 expression dose [1-10 µg/ml (EC50 0.5-1 µg/ml)] and time (24-72 h) dependently, significantly enhanced expression of ADAMTS4 mRNA, and promoted cartilage degeneration through collagen and proteoglycan degradation. Periostin induction of MMP-13 expression was inhibited by CCT031374 hydrobromide, an inhibitor of the canonical Wnt/ß-catenin signaling pathway. In addition, siRNA-mediated knockdown of endogenous periostin blocked constitutive MMP-13 expression. These findings implicate periostin as a catabolic protein that promotes cartilage degeneration in OA by up-regulating MMP-13 through canonical Wnt signaling.

Membrane-Type 1 Matrix Metalloproteinase Downregulates Fibroblast Growth Factor-2 Binding to the Cell Surface and Intracellular Signaling.

Membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14), a transmembrane proteinase with an extracellular catalytic domain and a short cytoplasmic tail, degrades extracellular matrix components and controls diverse cell functions through proteolytic and non-proteolytic interactions with extracellular, intracellular, and transmembrane proteins. Here we show that in tumor cells MT1-MMP downregulates fibroblast growth factor-2 (FGF-2) signaling by reducing the amount of FGF-2 bound to the cell surface with high and low affinity. FGF-2 induces weaker activation of ERK1/2 MAP kinase in MT1-MMP expressing cells than in cells devoid of MT1-MMP. This effect is abolished in cells that express proteolytically inactive MT1-MMP but persists in cells expressing MT1-MMP mutants devoid of hemopexin-like or cytoplasmic domain, showing that FGF-2 signaling is downregulated by MT1-MMP proteolytic activity. MT1-MMP expression results in downregulation of FGFR-1 and -4, and in decreased amount of cell surface-associated FGF-2. In addition, MT1-MMP strongly reduces the amount of FGF-2 bound to the cell surface with low affinity. Because FGF-2 association with low-affinity binding sites is a prerequisite for binding to its high-affinity receptors, downregulation of low-affinity binding to the cell surface results in decreased FGF-2 signaling. Consistent with this conclusion, FGF-2 induction of tumor cell migration and invasion in vitro is stronger in cells devoid of MT1- MMP than in MT1-MMP expressing cells. Thus, MT1-MMP controls FGF-2 signaling by a proteolytic mechanism that decreases the cell's biological response to FGF-2.

PKCε activation promotes FGF-2 exocytosis and induces endothelial cell proliferation and sprouting.

Protein kinase C epsilon (PKCε) activation controls fibroblast growth factor-2 (FGF-2) angiogenic signaling. Here, we examined the effect of activating PKCε on FGF-2 dependent vascular growth and endothelial activation. ψεRACK, a selective PKCε agonist induces pro-angiogenic responses in endothelial cells, including formation of capillary like structures and cell growth. These effects are mediated by FGF-2 export to the cell membrane, as documented by biotinylation and immunofluorescence, and FGF-2/FGFR1 signaling activation, as attested by ERK1/2-STAT-3 phosphorylation and de novo FGF-2 synthesis. Similarly, vascular endothelial growth factor (VEGF) activates PKCε in endothelial cells, and promotes FGF-2 export and FGF-2/FGFR1 signaling activation. ψεRACK fails to elicit responses in FGF-2(-/-) endothelial cells, and in cells pretreated with methylamine (MeNH2), an exocytosis inhibitor, indicating that both intracellular FGF-2 and its export toward the membrane are required for the ψεRACK activity. In vivo ψεRACK does not induce angiogenesis in the rabbit cornea. However, ψεRACK promotes VEGF angiogenic responses, an effect sustained by endothelial FGF-2 release and synthesis, since anti-FGF-2 antibody strongly attenuates VEGF responses. The results demonstrate that PKCε stimulation promotes angiogenesis and modulates VEGF activity, by inducing FGF-2 release and autocrine signaling.

TGF-ß1 induces endothelial cell apoptosis by shifting VEGF activation of p38(MAPK) from the prosurvival p38ß to proapoptotic p38α.

TGF-ß1 and VEGF, both angiogenesis inducers, have opposing effects on vascular endothelial cells. TGF-ß1 induces apoptosis; VEGF induces survival. We have previously shown that TGF-ß1 induces endothelial cell expression of VEGF, which mediates TGF-ß1 induction of apoptosis through activation of p38 mitogen-activated protein kinase (MAPK). Because VEGF activates p38(MAPK) but protects the cells from apoptosis, this finding suggested that TGF-ß1 converts p38(MAPK) signaling from prosurvival to proapoptotic. Four isoforms of p38(MAPK) -α, ß, γ, and δ-have been identified. Therefore, we hypothesized that different p38(MAPK) isoforms control endothelial cell apoptosis or survival, and that TGF-ß1 directs VEGF activation of p38(MAPK) from a prosurvival to a proapoptotic isoform. Here, we report that cultured endothelial cells express p38α, ß, and γ. VEGF activates p38ß, whereas TGF-ß1 activates p38α. TGF-ß1 treatment rapidly induces p38α activation and apoptosis. Subsequently, p38α activation is downregulated, p38ß is activated, and the surviving cells become refractory to TGF-ß1 induction of apoptosis and proliferate. Gene silencing of p38α blocks TGF-ß1 induction of apoptosis, whereas downregulation of p38ß or p38γ expression results in massive apoptosis. Thus, in endothelial cells p38α mediates apoptotic signaling, whereas p38ß and p38γ transduce survival signaling. TGF-ß1 activation of p38α is mediated by VEGF, which in the absence of TGF-ß1 activates p38ß. Therefore, these results show that TGF-ß1 induces endothelial cell apoptosis by shifting VEGF signaling from the prosurvival p38ß to the proapoptotic p38α.

Protein targets of inflammatory serine proteases and cardiovascular disease.

Serine proteases are a key component of the inflammatory response as they are discharged from activated leukocytes and mast cells or generated through the coagulation cascade. Their enzymatic activity plays a major role in the body's defense mechanisms but it has also an impact on vascular homeostasis and tissue remodeling. Here we focus on the biological role of serine proteases in the context of cardiovascular disease and their mechanism(s) of action in determining specific vascular and tissue phenotypes. Protease-activated receptors (PARs) mediate serine protease effects; however, these proteases also exert a number of biological activities independent of PARs as they target specific protein substrates implicated in vascular remodeling and the development of cardiovascular disease thus controlling their activities. In this review both PAR-dependent and -independent mechanisms of action of serine proteases are discussed for their relevance to vascular homeostasis and structural/functional alterations of the cardiovascular system. The elucidation of these mechanisms will lead to a better understanding of the molecular forces that control vascular and tissue homeostasis and to effective preventative and therapeutic approaches.

Correlation between plasma osteopontin levels and aortic valve calcification: potential insights into the pathogenesis of aortic valve calcification and stenosis.

OBJECTIVE: The inflammatory process of aortic stenosis involves the differentiation of aortic valve myofibroblasts into osteoblasts. Osteopontin, a proinflammatory glycoprotein, both stimulates differentiation of myofibroblasts and regulates the deposition of calcium by osteoblasts. Osteopontin levels are increased in patients with such conditions as end-stage renal disease, ectopic calcification, and autoimmune disease. We hypothesized that increased plasma osteopontin levels might be associated with the presence of aortic valve calcification and stenosis. METHODS: Venous blood from volunteers older than 65 years undergoing routine echocardiographic analysis or aortic valve surgery for aortic stenosis was collected. Plasma osteopontin levels were measured by means of enzyme-linked immunosorbent assay. The presence of aortic stenosis was defined as an aortic valve area of less than 2.0 cm(2). Aortic valve calcification was assessed by using a validated echocardiographic grading system (1, none; 2, mild; 3, moderate; 4, severe). Comparisons were performed with nonpaired t tests. RESULTS: Aortic stenosis was present in 23 patients (mean age, 78 years) and was absent in 7 patients (mean age, 72 years). Aortic valve calcification scores were 3.5 +/- 0.6 and 1.3 +/- 0.5 in patients with and without aortic stenosis, respectively (P < .001). Patients with no or mild aortic valve calcification had lower osteopontin levels compared with patients with moderate or severe aortic valve calcification (406.1 +/- 165.8 vs 629.5 +/- 227.5 ng/mL, P = .01). Similarly, patients with aortic stenosis had higher osteopontin levels compared with patients without aortic stenosis (652.2 +/- 218.7 vs 379.7 +/- 159.9 ng/mL, P < .01). CONCLUSION: Increased levels of plasma osteopontin are associated with the presence of aortic valve calcification and stenosis. These findings suggest that osteopontin might play a functional role in the pathogenesis of calcific aortic stenosis.

Inhibition of smooth muscle cell migration and neointima formation in vein grafts by overexpression of matrix metalloproteinase-3.

OBJECTIVE: Saphenous vein grafts suffer from neointima formation following bypass surgery. Matrix metalloproteinases (MMPs) play important roles in this process. We examined MMP-3 for its therapeutic potential to prevent smooth muscle cell migration and neointima formation in venous bypass grafts using adenovirus-mediated gene transfer. METHODS: Human aortic smooth muscle cells (HASMC) were transduced with adenoviral vectors encoding ss-galactosidase (Ad.ssgal) [corrected] or human MMP-3 (Ad.hMMP-(3)), [corrected] and characterized for migration in the amniotic membrane stroma as an in vitro model of the vascular wall. Cholesterol-fed New Zealand white rabbits underwent jugular vein bypass grafting into carotid arteries. Before insertion, grafts were incubated ex vivo with either Ad.ssgal [corrected] or hMMP-3. Transgene expression was characterized by immunohistochemistry and in situ zymography. Grafts (n = 6) were explanted after 28 days and intimal hyperplasia was quantified. RESULTS: Migration of HASMC was significantly reduced when transduced with Ad.hMMP-(3) [corrected] compared to controls (P < .001). Immunocytochemistry of Ad.hMMP-(3) [corrected] transduced venous grafts localized this protein to the intima. In situ-zymography showed increased MMP activity in the intima of Ad.hMMP-(3) [corrected] transfected grafts. Stenosis degree (P = .001), intima/media-ratio (P = .023) and lesion thickness (P = .003) were significantly reduced in grafts transduced with Ad.MMP-3 in comparison to controls. There was no difference inside control groups. CONCLUSION: MMP-3 overexpression inhibits formation of intimal hyperplasia in arterialized vein grafts. Adenovirus mediated gene transfer of MMP-3 may be of clinical use to prevent vein graft stenosis following bypass surgery.

Transforming growth factor-beta 1 (TGF-beta1) induces angiogenesis through vascular endothelial growth factor (VEGF)-mediated apoptosis.

VEGF and TGF-beta1 induce angiogenesis but have opposing effects on endothelial cells. VEGF protects endothelial cells from apoptosis; TGF-beta1 induces apoptosis. We have previously shown that VEGF/VEGF receptor-2 (VEGFR2) signaling mediates TGF-beta1 induction of apoptosis. This finding raised an important question: Does this mechanism stimulate or inhibit angiogenesis? Here we report that VEGF-mediated apoptosis is required for TGF-beta1 induction of angiogenesis. In vitro the apoptotic effect of TGF-beta1 on endothelial cells is rapid and followed by a long period in which the cells are refractory to apoptosis induction by TGF-beta1. Inhibition of VEGF/VEGFR2 signaling abrogates formation of cord-like structures by TGF-beta1 with an effect comparable to that of z-VAD, an apoptosis inhibitor. Similarly, genetic deficiency of VEGF abolishes TGF-beta1 upregulation of endothelial cell differentiation and formation of vascular structures in embryoid bodies. In vivo TGF-beta1 induces endothelial cell apoptosis as rapidly as in vitro. Inhibition of VEGF blocks TGF-beta1 induction of both apoptosis and angiogenesis, an effect similar to that of z-VAD. Thus, TGF-beta1 induction of angiogenesis requires a rapid and transient apoptotic effect mediated by VEGF/VEGFR2. This novel, unexpected role of VEGF and VEGFR2 indicates VEGF-mediated apoptosis as a potential target to control angiogenesis.

Topical mitogen-activated protein kinases inhibition reduces intimal hyperplasia in arterialized vein grafts.

OBJECTIVE: Vein graft arterialization results in activation of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinases-1 and -2 (ERK1/2), which have been implicated in cell proliferation, migration, and apoptosis. The goal of our study was to characterize the effect of MAPK inhibition on intimal hyperplasia (IH) in arterialized vein grafts in hypercholesterolemic rabbits. METHODS: Reversed bilateral jugular vein to common carotid artery interposition grafts were constructed in 16 New Zealand White rabbits. The veins were incubated for 30 min prior to grafting with either the synthetic ERK1/2 activation inhibitor UO126 or the control vehicle. Vein graft and control jugular vein were harvested 3 h, 1 d, and 28 d after arterialization for histological and biochemical analyses. RESULTS: Treatment with UO126 was associated with 31% reduction in mean intimal area (1.68 +/- 0.78 mm(2)versus 2.44 +/- 1.65 mm(2); mean +/- SD; P = 0.036) relative to controls. The intima-to-media ratio of UO126-treated vein grafts decreased by 29% (0.53 +/- 0.04 versus 0.74 +/- 0.06; mean +/- SD; P < 0.01) compared to controls, vehicle-treated vein grafts. There was also significant increase in apoptosis in UO126-treated vein graft medial cell layer at 1 d. CONCLUSION: Topical administration of UO126 before vein grafting significantly decreases IH in arterialized vein grafts in hypercholesterolemic rabbits. These results may have significant implications for the development of strategies aimed at blocking or reducing IH in bypass grafts. Therefore, further evaluation of this simple strategy to improve vein graft patency following coronary artery or peripheral vascular bypass surgery is warranted.

TGF-beta1 induces rearrangement of FLK-1-VE-cadherin-beta-catenin complex at the adherens junction through VEGF-mediated signaling.

VEGF and TGF-beta1 induce angiogenesis but have opposing effects on vascular endothelial cells: VEGF promotes survival; TGF-beta1 induces apoptosis. We have previously shown that TGF-beta1 induces endothelial cell apoptosis via up-regulation of VEGF expression and activation of signaling through VEGF receptor-2 (flk-1). In context with TGF-beta1, VEGF signaling is transiently converted from a survival into an apoptotic one. VEGF promotes cell survival in part via activation of PI3K/Akt by a mechanism dependent on the formation of a multi-protein complex that includes flk-1 and the adherens junction proteins VE-cadherin and beta-catenin. Here we report that TGF-beta1 induces rearrangement of the adherens junction complex by separating flk-1 from VE-cadherin and increasing beta-catenin association with both flk-1 and VE-cadherin. This rearrangement is caused neither by changes in adherens junction mRNA or protein expression nor by post-translational modification, and requires VEGF signaling through flk-1. These results show that the adherens junction is an important regulatory component of TGF-beta1-VEGF interaction in endothelial cells.