C57BL/6 NK cell gene complex is crucially involved in vascular remodeling.

OBJECTIVE: NK cells are known to be involved in cardiovascular disease processes. One of these processes, vascular remodeling, may strongly differ between individuals and mouse strains such as the C57BL/6 and BALB/c. Moreover, C57BL/6 and BALB/c mice vary in immune responses and in the composition of their Natural Killer gene Complex (NKC). Here we study the role of NK cells, and in particular the C57BL/6 NKC in vascular remodeling and intimal hyperplasia formation. METHODS AND RESULTS: C57BL/6, BALB/c and CMV1(r) mice, a BALB/c strain congenic for the C57BL/6 NKC, were used in an injury induced cuff model and a vein graft model. NK cell depleted C57BL/6 mice demonstrated a 43% reduction in intimal hyperplasia after femoral artery cuff placement compared to control C57BL/6 mice (p<0.05). Cuff placement and vein grafting resulted in profound intimal hyperplasia in C57BL/6 mice, but also in CMV1(r) mice, whereas this was significantly less in BALB/c mice. Significant more leukocyte infiltrations and IFN-γ staining were seen in both C57BL/6 and CMV1(r) vein grafts compared to BALB/c vein grafts. CONCLUSIONS: These data demonstrate an important role for NK cells in intimal hyperplasia and vascular remodeling. Furthermore, the C57BL/6 NKC in CMV1(r) mice stimulates vascular remodeling most likely through the activation of (IFN-γ-secreting) NK-cells that modulate the outcome of vascular remodeling.

Human platelet lysate as a fetal bovine serum substitute improves human adipose-derived stromal cell culture for future cardiac repair applications.

Adipose-derived stromal cells (ASC) are promising candidates for cell therapy, for example to treat myocardial infarction. Commonly, fetal bovine serum (FBS) is used in ASC culturing. However, FBS has several disadvantages. Its effects differ between batches and, when applied clinically, transmission of pathogens and antibody development against FBS are possible. In this study, we investigated whether FBS can be substituted by human platelet lysate (PL) in ASC culture, without affecting functional capacities particularly important for cardiac repair application of ASC. We found that PL-cultured ASC had a significant 3-fold increased proliferation rate and a significantly higher attachment to tissue culture plastic as well as to endothelial cells compared with FBS-cultured ASC. PL-cultured ASC remained a significant 25% smaller than FBS-cultured ASC. Both showed a comparable surface marker profile, with the exception of significantly higher levels of CD73, CD90, and CD166 on PL-cultured ASC. PL-cultured ASC showed a significantly higher migration rate compared with FBS-cultured ASC in a transwell assay. Finally, FBS- and PL-cultured ASC had a similar high capacity to differentiate towards cardiomyocytes. In conclusion, this study showed that culturing ASC is more favorable in PL-supplemented medium compared with FBS-supplemented medium.

CD133+ circulating haematopoietic progenitor cells predict for response to sorafenib plus erlotinib in non-small cell lung cancer patients.

BACKGROUND: Blood-based biomarkers may be particularly useful for patient selection and prediction of treatment response for angiogenesis inhibitors. Circulating endothelial cells (CECs) and haematopoietic progenitor cells (HPCs) might have a role in tumour angiogenesis and in tumour growth. Measurement of CECs and HPCs in the blood of patients could be a simple, non-invasive way to monitor or predict responses to treatment. METHODS: (VEGFR2(+)) CECs(,) (CD133(+)) HPCs, plasma vascular endothelial growth factor (VEGF) and erythropoietin were measured in blood from 25 non-small cell lung cancer (NSCLC) patients before and during treatment with sorafenib plus erlotinib (SO/ER). In order to assess the drug specificity of changes in CECs and HPCs, 18 patients treated with bevacizumab plus erlotinib (BV/ER) and 10 patients with erlotinib (ER) monotherapy were studied. Response was measured in all patient groups by Response Evaluation Criteria in Solid Tumors (RECIST). RESULTS: At day 7, SO/ER-treated patients showed a three-fold increase in CECs (P<0.0001) comparable to BV/ER-treated patients (P<0.01), and the CECs did not change with erlotinib treatment (P=0.8). At day 7, CD133(+)/HPCs decreased with SO/ER treatment (P<0.0001). HPC numbers did not change with either BV/ER or erlotinib. In SO/ER-treated patients pre-treatment CD133(+)/HPCs were significantly lower in responders (P=0.01) and pre-treatment CD133(+)/HPC numbers lower than the median correlated with a longer time-to-progression (TTP) (P=0.037). CONCLUSION: Pre-treatment CD133(+)/HPCs are a promising candidate biomarker to further explore for use in selecting NSCLC patients who might benefit from SO/ER treatment.

Fasudil reduces monocrotaline-induced pulmonary arterial hypertension: comparison with bosentan and sildenafil.

Pulmonary arterial hypertension (PAH) still cannot be cured, warranting the search for novel treatments. Fasudil (a Rho kinase inhibitor) was compared with bosentan (an endothelin receptor blocker) and sildenafil (a phosphodiesterase 5 inhibitor), with emphasis on right ventricular (RV) function, in a reversal rat model of monocrotaline (MCT)-induced PAH. In addition, the effects of combining bosentan or sildenafil with fasudil were studied. MCT (40 mg·kg body weight(-1)) induced clear PAH in male Wistar rats (n = 9). After 28 days, echocardiography, RV catheterisation and histochemistry showed that cardiac frequency, stroke volume and RV contractility had deteriorated, accompanied by RV dilatation and hypertrophy, and marked pulmonary arterial wall thickening. Mean pulmonary arterial pressure and pulmonary vascular resistance increased significantly compared to healthy rats (n = 9). After 14 days, MCT-treated rats received a 14-day oral treatment with bosentan, sildenafil, fasudil or a combination of fasudil with either bosentan or sildenafil (all n = 9). All treatments preserved cardiac frequency, stroke volume and RV contractility, and reduced pulmonary vascular resistance and RV dilatation. Fasudil lowered RV systolic pressure and mean pulmonary arterial pressure significantly, by reducing pulmonary arterial remodelling, which reduced RV hypertrophy. Combining bosentan or sildenafil with fasudil had no synergistic effect. Fasudil significantly improved PAH, to a greater degree than did bosentan and sildenafil.

Activated complement is more extensively present in diseased aortic valves than naturally occurring complement inhibitors: a sign of ongoing inflammation.

BACKGROUND: Recent studies indicate a role for complement in the pathogenesis of aortic valve disease. However, the role of naturally occurring anti-complement mediators in this context is unknown. In this study, we have analysed this in three different pathological conditions of the aortic valve: degeneration, atherosclerosis and bacterial endocarditis. MATERIALS AND METHODS: Human aortic valves were obtained at autopsy (n = 30): 5 control valves, 10 aortic valves with atherosclerotic changes, 10 aortic valves with degenerative changes and 5 degenerative changed aortic valves with bacterial infection. These valves were analysed immunohistochemically for the presence of activated complement (C3d and C5b9) and the complement inhibitors C1-inh and clusterin. Areas of positivity were then quantified. RESULTS: C3d, C5b9 and the complement inhibitors C1-inh and clusterin depositions were mainly found in the endothelium and extracellular matrix in aortic valves. All these mediators were already present in control valves, but the area of positivity increased significantly in response to the different diseases, with the highest increase in response to bacterial endocarditis. Interestingly, in all three aortic diseases, the depositions of complement were significantly more widespread than that of their inhibitors. CONCLUSIONS: Our study indicates that anti-complement mediators (C1-inh and clusterin) are deposited in diseased aortic valves together with activated complement, indicating an existing counter response against complement locally in the valve. However, deposition of activated complement is significantly more widespread than that of its inhibitors, which could explain ongoing inflammation in those diseased aortic valves.

Decidual vascularization and the expression of angiogenic growth factors and proteases in first trimester spontaneous abortions.

BACKGROUND: Decidual vascular development is important for implantation. This study analysed decidual vascular adaptation to implantation in correlation with miscarriage in decidual secretory endometrium (DSE), decidua parietalis (DP) and decidua basalis (DB) of miscarriage patients and matched controls. METHODS: Decidua was obtained during first trimester termination of pregnancy (controls) and vacuum aspiration in case of missed abortion (cases). Vascularization and the expression of VEGF-A, placental growth factor, Flt-1, KDR, angiopoietin (Ang)-1, Ang-2, TIE-2, and membrane-type matrix metalloproteinases MT1-, MT2-, MT3- and MT5-MMP were determined at mRNA and protein level. Uterine natural killer cells (CD56), macrophages (CD68), proliferation (Ki67) and apoptosis (activated caspase-3) were evaluated in consecutive sections. RESULTS: Decidual vascularization showed differences between cases and controls, i.e. fewer vessels with larger circumference in cases. This correlated with the differential expressions of various factors at mRNA/antigen level and with increased endothelial flt1, KDR, MT2- and MT5-MMP expression in miscarriage patients. The differences between cases and controls were probably not based on altered proliferation and/or apoptosis, since Ki67 and active Caspase-3 showed comparable expression levels in both groups. Although DB of cases and controls showed similar amounts of CD56- and CD68-positive cells, the case group did show elevated levels of CD56 in DSE (P < 0.05) and of CD68 in DP compared with the control group (P < 0.05). CONCLUSIONS: The differences in vascularization and in the expression of angiogenic factors and proteases between groups suggest a correlation between decidual vascularization and the occurrence of miscarriages.

Cooperative effect of TNFalpha, bFGF, and VEGF on the formation of tubular structures of human microvascular endothelial cells in a fibrin matrix. Role of urokinase activity.

In angiogenesis associated with tissue repair and disease, fibrin and inflammatory mediators are often involved. We have used three-dimensional fibrin matrices to investigate the humoral requirements of human microvascular endothelial cells (hMVEC) to form capillary-like tubular structures. bFGF and VEGF165 were unable to induce tubular structures by themselves. Simultaneous addition of one or both of these factors with TNFalpha induced outgrowth of tubules, the effect being the strongest when bFGF, VEGF165, and TNFalpha were added simultaneously. Exogenously added u-PA, but not its nonproteolytic amino-terminal fragment, could replace TNFalpha, suggesting that TNFalpha-induced u-PA synthesis was involved. Soluble u-PA receptor (u-PAR) or antibodies that inhibited u-PA activity prevented the formation of tubular structures by 59-99%. epsilon-ACA and trasylol which inhibit the formation and activity of plasmin reduced the extent of tube formation by 71-95%. TNFalpha or u-PA did not induce tubular structures without additional growth factors. bFGF and VEGF165 enhanced of the u-PAR by 72 and 46%, but TNFalpha itself also increased u-PAR in hMVEC by 30%. Induction of mitogenesis was not the major contribution of bFGF and VEGF165 because the cell number did not change significantly in the presence of TNFalpha, and tyrphostin A47, which inhibited mitosis completely, reduced the formation of tubular structures only by 28-36%. These data show that induction of cell-bound u-PA activity by the cytokine TNFalpha is required in addition to the angiogenic factors VEGF165 and/or bFGF to induce in vitro formation of capillary-like structures by hMVEC in fibrin matrices. These data may provide insight in the mechanism of angiogenesis as occurs in pathological conditions.

Angiopoietin-2, permeability oedema, occurrence and severity of ALI/ARDS in septic and non-septic critically ill patients.

BACKGROUND: Angiopoietin-2 and vascular endothelial growth factor (VEGF) may impair vascular barrier function while angiopoietin-1 may protect it. It was hypothesised that circulating angiopoietin-2 is associated with pulmonary permeability oedema and severity of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) during septic or non-septic critical illness. METHODS: Plasma levels of angiopoietin-1 and angiopoietin-2 were measured in mechanically ventilated patients (24 with sepsis, 88 without sepsis), together with the pulmonary leak index (PLI) for 67-gallium-labelled transferrin and extravascular lung water (EVLW) by transpulmonary thermal-dye dilution as measures of pulmonary permeability and oedema, respectively. ALI/ARDS was characterised by consensus criteria and the lung injury score (LIS). Plasma VEGF and von Willebrand factor (VWF) levels were assayed. RESULTS: Angiopoietin-2, VWF, PLI, EVLW and LIS were higher in patients with sepsis than in those without sepsis and higher in patients with ALI/ARDS (n = 10/12 in sepsis, n = 19/8 in non-sepsis) than in those without. VEGF was also higher in patients with sepsis than in those without. Patients with high PLI, regardless of EVLW, had higher angiopoietin-2 levels than patients with normal PLI and EVLW. Angiopoietin-2 correlated with the PLI, LIS and VWF levels (minimum r = 0.34, p<0.001) but not with EVLW. Angiopoietin-2 and VWF were predictive for ARDS in receiver operating characteristic curves (minimum area under the curve = 0.69, p = 0.006). Angiopoietin-1 and VEGF did not relate to the permeability oedema of ALI/ARDS. CONCLUSION: Circulating angiopoietin-2 is associated with pulmonary permeability oedema, occurrence and severity of ALI/ARDS in patients with and without sepsis. The correlation of angiopoietin-2 with VWF suggests activated endothelium as a common source.

Increased infiltration of Chlamydophila pneumoniae in the vessel wall of human veins after perfusion.

BACKGROUND: Several studies have suggested an association between Chlamydophila pneumoniae (Cp) infection and atherosclerosis. A recent study detected Cp DNA in the saphenous vein of 12% of all patients before bypass grafting and in 38% of failed grafts. We used a system in which human veins were perfused with autologous blood under arterial pressure. MATERIALS AND METHODS: Veins were surplus segments of saphenous veins of coronary artery bypass grafting (CABG) patients. Vein grafts were perfused with the blood of the same patient after CABG procedures. Veins were analysed for Cp-specific membrane protein using immunohistochemical and PCR analysis. Veins were analysed before and after perfusion (up to 4 h). The number of Cp positive cells was then quantified in the vein layers. RESULTS: Cp protein was detected within macrophages only. In non-perfused veins, Cp was present in the adventitia in 91% of all patients, in the circular (64%) and longitudinal (23%) layer of the media. No positivity was found in the intima. Perfusion subsequently resulted in a significant increase of Cp positive cells within the circular layer of the media that, however, differed strongly between different patients. Cp DNA was not detected by PCR in those specimens. CONCLUSION: Cp protein was present in 91% of veins, but the number of positive cells differed remarkably between patients. Perfusion of veins resulted in increased infiltration of Cp into the circular layer. These results may point to a putative discriminating role of Cp with respect to graft failure between different patients.

Involvement of Rho kinase in endothelial barrier maintenance.

OBJECTIVE: Rho kinase mediates vascular leakage caused by many vasoactive agents including thrombin. Enhanced Rho kinase activity induces endothelial barrier dysfunction by a contractile mechanism via inactivation of Myosin Phosphatase (MP). Here, we investigated the contribution of basal Rho kinase activity to the regulation of endothelial barrier integrity. METHODS AND RESULTS: Using a phospho-specific antibody against the myosin phosphatase targeting subunit (Thr696-MYPT1) as a marker for Rho kinase activity, basal endothelial Rho kinase activity was observed at cell-cell contact sites, in vitro and in situ. Thrombin enhanced MYPT phosphorylation at F-actin stress fibers. Inhibition of basal Rho kinase activity for 24 hours or depletion of Rho kinase (ROCK-I and -II) by siRNA disrupted endothelial barrier integrity, opposite to the previously observed protection from the thrombin-enhanced endothelial permeability. This barrier dysfunction could not be explained by changes in RhoA, Rac1, eNOS, or apoptosis. Remarkably, basal Rho kinase activity was essential for proper expression of the adhesion molecule VE-cadherin. CONCLUSIONS: Rho kinase has opposing activities in regulation of endothelial barrier function: (1) an intrinsic barrier-protective activity at the cell margins, and (2) an induced barrier-disruptive activity at contractile F-actin stress fibers. These findings may have implications for long-term antivascular leak therapy.

Natural killer cells and CD4+ T-cells modulate collateral artery development.

OBJECTIVE: The immune system is thought to play a crucial role in regulating collateral circulation (arteriogenesis), a vital compensatory mechanism in patients with arterial obstructive disease. Here, we studied the role of lymphocytes in a murine model of hindlimb ischemia. METHODS AND RESULTS: Lymphocytes, detected with markers for NK1.1, CD3, and CD4, invaded the collateral vessel wall. Arteriogenesis was impaired in C57BL/6 mice depleted for Natural Killer (NK)-cells by anti-NK1.1 antibodies and in NK-cell-deficient transgenic mice. Arteriogenesis was, however, unaffected in J alpha281-knockout mice that lack NK1.1+ Natural Killer T (NKT)-cells, indicating that NK-cells, rather than NKT-cells, are involved in arteriogenesis. Furthermore, arteriogenesis was impaired in C57BL/6 mice depleted for CD4+ T-lymphocytes by anti-CD4 antibodies, and in major histocompatibility complex (MHC)-class-II-deficient mice that more selectively lack mature peripheral CD4+ T-lymphocytes. This impairment was even more profound in anti-NK1.1-treated MHC-class-II-deficient mice that lack both NK- and CD4+ T-lymphocytes. Finally, collateral growth was severely reduced in BALB/c as compared with C57BL/6 mice, 2 strains with different bias in immune responsiveness. CONCLUSIONS: These data show that both NK-cells and CD4+ T-cells modulate arteriogenesis. Promoting lymphocyte activation may represent a promising method to treat ischemic disease.

Vascular growth in ischemic limbs: a review of mechanisms and possible therapeutic stimulation.

Stimulation of vascular growth to treat limb ischemia is promising, and early results obtained from uncontrolled clinical trials using angiogenic agents, e.g., vascular endothelial growth factor, led to high expectations. However, negative results from recent placebo-controlled trials warrant further research. Here, current insights into mechanisms of vascular growth in the adult, in particular the role of angiogenic factors, the immune system, and bone marrow, were reviewed, together with modes of its therapeutic stimulation and results from recent clinical trials. Three concepts of vascular growth have been described to date-angiogenesis, vasculogenesis, and arteriogenesis (collateral artery growth)-which represent different aspects of an integrated process. Stimulation of arteriogenesis seems clinically most relevant and has most recently been attempted using autologous bone marrow transplantation with some beneficial results, although the mechanism of action is not completely understood. Better understanding of the highly complex molecular and cellular mechanisms of vascular growth may yet lead to meaningful clinical applications.

The minor histocompatibility antigen 1 (HMHA1)/ArhGAP45 is a RacGAP and a novel regulator of endothelial integrity.

Endothelial cells line the vasculature and act as gatekeepers that control the passage of plasma, macromolecules and cells from the circulation to the interstitial space. Dysfunction of the endothelial barrier can lead to uncontrolled leak or edema. Vascular leakage is a hallmark of a range of diseases and despite its large impact no specialized therapies are available to prevent or reduce it. RhoGTPases are known key regulators of cellular behavior that are directly involved in the regulation of the endothelial barrier. We recently performed a comprehensive analysis of the effect of all RhoGTPases and their regulators on basal endothelial integrity. In addition to novel positive regulators of endothelial barrier function, we also identified novel negative regulators, of which the ArhGAP45 (also known as HMHA1) was the most significant. We now demonstrate that ArhGAP45 acts as a Rac-GAP (GTPase-Activating Protein) in endothelial cells, which explains its negative effect on endothelial barrier function. Silencing ArhGAP45 not only promotes basal endothelial barrier function, but also increases cellular surface area and induces sprout formation in a 3D-fibrin matrix. Our data further shows that loss of ArhGAP45 promotes migration and shear stress adaptation. In conclusion, we identify ArhGAP45 (HMHA1) as a novel regulator, which contributes to the fine-tuning of the regulation of basal endothelial integrity.

N(epsilon)-(carboxymethyl)lysine depositions in intramyocardial blood vessels in human and rat acute myocardial infarction: a predictor or reflection of infarction?

OBJECTIVE: Advanced glycation end products (AGEs), such as N(epsilon)-(carboxymethyl)lysine (CML), are implicated in vascular disease. We previously reported increased CML accumulation in small intramyocardial blood vessels in diabetes patients. Diabetes patients have an increased risk for acute myocardial infarction (AMI). Here, we examined a putative relationship between CML and AMI. METHODS AND RESULTS: Heart tissue was stained for CML, myeloperoxidase, and E-selectin in AMI patients (n=26), myocarditis patients (n=17), and control patients (n=15). In AMI patients, CML depositions were 3-fold increased compared with controls in the small intramyocardial blood vessels and predominantly colocalized with activated endothelium (E-selectin-positive) both in infarction and noninfarction areas. A trend of increased CML positivity of the intima of epicardial coronary arteries did not reach significance in AMI patients. In the rat heart AMI model, CML depositions were undetectable after 24 hours of reperfusion, but became clearly visible after 5 days of reperfusion. In line with an inflammatory contribution, human myocarditis was also accompanied by accumulation of CML on the endothelium of intramyocardial blood vessels. CONCLUSIONS: CML, present predominantly on activated endothelium in small intramyocardial blood vessels in patients with AMI, might reflect an increased risk for AMI rather than being a result of AMI.

Exercise effects on perivascular adipose tissue: endocrine and paracrine determinants of vascular function.

Obesity is a global epidemic, accompanied by increased risk of type 2 diabetes and cardiovascular disease. Adipose tissue hypertrophy is associated with adipose tissue inflammation, which alters the secretion of adipose tissue-derived bioactive products, known as adipokines. Adipokines determine vessel wall properties such as smooth muscle tone and vessel wall inflammation. Exercise is a mainstay of prevention of chronic, non-communicable diseases, type 2 diabetes and cardiovascular disease in particular. Aside from reducing adipose tissue mass, exercise has been shown to reduce inflammatory activity in this tissue. Mechanistically, contracting muscles release bioactive molecules known as myokines, which alter the metabolic phenotype of adipose tissue. In adipose tissue, myokines induce browning, enhance fatty acid oxidation and improve insulin sensitivity. In the past years, the perivascular adipose tissue (PVAT) which surrounds the vasculature, has been shown to control vascular tone and inflammation through local release of adipokines. In obesity, an increase in mass and inflammation of PVAT culminate in dysregulation of adipokine secretion, which contributes to vascular dysfunction. This review describes our current understanding of the mechanisms by which active muscles interact with adipose tissue and improve vascular function. Aside from the exercise-dependent regulation of canonical adipose tissue function, we will focus on the interactions between skeletal muscle and PVAT and the role of novel myokines, such as IL-15, FGF21 and irisin, in these interactions. LINKED ARTICLES: This article is part of a themed section on Molecular Mechanisms Regulating Perivascular Adipose Tissue - Potential Pharmacological Targets? To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.20/issuetoc.

A CDC42-centered signaling unit is a dominant positive regulator of endothelial integrity.

Endothelial barrier function is carefully controlled to protect tissues from edema and damage inflicted by extravasated leukocytes. RhoGTPases, in conjunction with myriad regulatory proteins, exert both positive and negative effects on the endothelial barrier integrity. Precise knowledge about the relevant mechanisms is currently fragmented and we therefore performed a comprehensive analysis of endothelial barrier regulation by RhoGTPases and their regulators. Combining RNAi with electrical impedance measurements we quantified the relevance of 270 Rho-associated genes for endothelial barrier function. Statistical analysis identified 10 targets of which six promoted- and four reduced endothelial barrier function upon downregulation. We analyzed in more detail two of these which were not previously identified as regulators of endothelial integrity. We found that the Rac1-GEF (Guanine nucleotide Exchange Factor) TIAM2 is a positive regulator and the Cdc42(Rac1)-GAP (GTPase-Activating Protein) SYDE1 is a negative regulator of the endothelial barrier function. Finally, we found that the GAP SYDE1 is part of a Cdc42-centered signaling unit, also comprising the Cdc42-GEF FARP1 and the Cdc42 effector PAK7 which controls the integrity of the endothelial barrier. In conclusion, using a siRNA-based screen, we identified new regulators of barrier function and found that Cdc42 is a dominant positive regulator of endothelial integrity.

Molecular weight fibrinogen variants determine angiogenesis rate in a fibrin matrix in vitro and in vivo.

BACKGROUND: During wound repair, fibrin acts both as a barrier to prevent blood loss and as a temporary matrix for the invasion and ingrowth of endothelial and tissue cells. A well-controlled angiogenesis process in the fibrinous exudate matrix is crucial for optimal wound healing. The composition and structure of the fibrin matrix are important determinants of the invasion of endothelial cells and capillary-tube formation into the matrix. OBJECTIVE: Fibrinogen circulates in a high and low molecular weight form (HMW and LMW, respectively) and the purpose of this study was to investigate how fibrin matrices from these naturally occurring fibrinogen variants influence angiogenesis. Angiogenesis was studied using an in vitro model in which human microvascular endothelial cells (hMVEC) were cultured on three-dimensional fibrin matrices from different fibrinogen forms, and using two in vivo mouse models. RESULTS: The in vitro angiogenesis in an HMW-fibrin matrix shows increased cell and tubular structure ingrowth compared with unfractionated fibrin matrix (median increase 58%, range 46-234%). The ingrowth of tubular structures in an LMW-fibrin matrices is decreased when compared with unfractionated fibrin (median decrease 70%, range 67-100%). Similar results were observed for in vivo angiogenesis. CONCLUSIONS: The naturally occurring fibrinogen variants HMW- and LMW-fibrin modulate the angiogenic capacity of endothelial cells in fibrin matrices. The different effects of the molecular weight fibrinogen variants provide further insight in the matrix characteristics in angiogenesis and could possibly be applied in the context of tissue engineering and wound healing.

Accelerated atherosclerosis and calcification in vein grafts: a study in APOE*3 Leiden transgenic mice.

Vein grafts fail due to development of intimal hyperplasia and accelerated atherosclerosis. Many murine genetic models in which genes are overexpressed, deleted, or mutated have been introduced recently. Therefore, mouse models are very well suited to dissect the relative contribution of different genes in the development of accelerated atherosclerosis. In the present study, we evaluated whether accelerated atherosclerosis in human vein grafts could be mimicked in hypercholesterolemic APOE*3 Leiden transgenic mice. Venous bypass grafting was performed in the carotid artery in APOE*3 Leiden mice fed either a standard chow diet or a high cholesterol-rich diet for 4 weeks. At several time points (0 hour to 28 days), mice were euthanized and the morphology of the vein grafts was analyzed. In normocholesterolemic mice, vein graft thickening up to 10-fold original thickness, predominantly consisting of alpha-smooth muscle cell actin-positive cells, was observed after 28 days. In hypercholesterolemic mice, accelerated atherosclerosis with accumulation of lipid-loaded foam cells was observed within 7 days after surgery. This accelerated atherosclerosis progressed in time and resulted in significant increase in vein graft thickening up to 50 times original thickness with foam cell-rich lesions and calcification within 28 days after surgery. The atherosclerotic lesions observed in these murine grafts show high morphological resemblance with the atherosclerotic lesions observed in human vein grafts. This accelerated, diet-dependent induction of atherosclerotic-like lesions in murine vein grafts provides a valuable tool in evaluating the mechanisms of accelerated atherosclerosis and therapeutic interventions of vein graft disease.

Gene transfer of the urokinase-type plasminogen activator receptor-targeted matrix metalloproteinase inhibitor TIMP-1.ATF suppresses neointima formation more efficiently than tissue inhibitor of metalloproteinase-1.

Proteases of the plasminogen activator (PA) and matrix metalloproteinase (MMP) system play an important role in smooth muscle cell (SMC) migration and neointima formation after vascular injury. Inhibition of either PAs or MMPs has previously been shown to result in decreased neointima formation in vivo. To inhibit both protease systems simultaneously, a novel hybrid protein, TIMP-1.ATF, was constructed consisting of the tissue inhibitor of metalloproteinase-1 (TIMP-1) domain, as MMP inhibitor, linked to the receptor-binding amino terminal fragment (ATF) of urokinase. By binding to the u-PA receptor this protein will not only anchor the TIMP-1 moiety directly to the cell surface, it will also prevent the local activation of plasminogen by blocking the binding of urokinase-type plasminogen activator (u-PA) to its receptor. Adenoviral expression of TIMP-1.ATF was used to inhibit SMC migration and neointima formation in human saphenous vein segments in vitro. SMC migration was inhibited by 65% in Ad.TIMP-1.ATF-infected cells. Infection with adenoviral vectors encoding the individual domains, Ad.TIMP-1 and Ad.ATF, reduced migration by 32% and 52%, respectively. Neointima formation in saphenous vein organ cultures infected with Ad.TIMP-1.ATF was inhibited by 72% compared with 42% reduction after Ad.TIMP-1 infection and 34% after Ad.ATF infection. These data show that binding of TIMP-1.ATF hybrid protein to the u-PA receptor at the cell surface strongly enhances the inhibitory effect of TIMP-1 on neointima formation in human saphenous vein cultures.

Unfractionated and low molecular weight heparin affect fibrin structure and angiogenesis in vitro.

Cancer patients treated for venous thromboembolism with low molecular weight heparin (LMWH) have a better survival rate than patients treated with unfractionated heparin (UFH). Because fibrin-associated angiogenesis is an important determinant in the progression and metastasis of many solid tumors, the effects of heparins on in vitro angiogenesis were investigated. Both UFH and LMWH inhibited bFGF-induced proliferation of human microvascular endothelial cells (hMVECs) to the same the extent (36-60%). VEGF165-induced proliferation was inhibited to a to a lesser extent (19-33%). Turbidity measurements and electron microscopy showed that the presence of LMWH during polymerization of the fibrin matrix led to a more transparent rigid network with thin fibrin bundles, whereas the presence of UFH resulted in a more opaque more porous network with thick fibrin fibers. We used a human in vitro angiogenesis model, which consisted of hMVECs seeded on top of a fibrin matrix, and stimulated the cells with basic fibroblast growth factor plus tumor necrosis factor a to induce capillary-like tubular structures. The formation of capillary-like tubular structures was retarded with matrices polymerized in the presence of LMWH (46% inhibition compared with a control matrix for both 1.5 and 10 units/ml LMWH), whereas matrices polymerized in the presence of UFH facilitated tubular structure formation (72 and 36% stimulation compared with a control matrix for 1.5 and 10 units/ml UFH, respectively). Similar results were obtained for cells stimulated with vascular endothelial growth factor plus tumor necrosis factor alpha. These data demonstrate the inhibitory effect of heparins on proliferation of hMVECs and provide a novel mechanism by which LMWH may affect tumor progression, namely reduced ingrowth of microvascular structures in a fibrinous stroma matrix by rendering it less permissive for invasion.