Characterization of lymphatic malformations using primary cells and tissue transcriptomes.

Lymphatic malformations (LMs) are disfiguring congenital anomalies characterized by aberrant growth of lymphatic vessels. They are broadly categorized histopathologically as macrocystic and microcystic. Although sclerotherapy has shown some success in the treatment of macrocystic malformations, there has been less progress with developing treatment strategies for microcystic malformations. In this study, we characterized lymphatic endothelial cells isolated from lymphatic and lymphaticovenous malformations. When compared to cells from normal lymphatic vessels, we found that the primary cultured malformed cells are morphologically different and also exhibited differences in binding, proliferation, migration and tube formation. Transcriptome analysis identified several genes whose expression was substantially higher in malformed compared to normal lymphatic endothelium, including DIRAS3 and FOXF1. Further analysis of LM tissue samples revealed distinguishing gene expression patterns that could pave the way to understanding the molecular pathogenesis of LMs. Based on gene expression signatures, we propose a new hypothesis that the subtype of localized LMs could be formed because of disruptions in lymph node development.

Comparison of a high throughput day case atrial fibrillation ablation service in a local hospital with standard regional tertiary cardiac centre care.

AIMS: We investigated safety and efficacy of a high throughput atrial fibrillation (AF) ablation service within a local non-cardiac centre compared with matched patients at the regional tertiary cardiac centre. METHODS AND RESULTS: Patients were consented in clinic and pre-assessed by clerical staff. Locally, other than physicians, staff had no experience of ablation and were trained with simulations. Pulmonary vein isolation (PVI) was performed with conscious sedation, on uninterrupted anticoagulation for at least 4 weeks. No transoesophageal echocardiogram (TOE) was performed. A 28 mm Arctic Front Advance Cryoballoon was used. A 20 mm Achieve wire demonstrated PVI on a portable laptop-based EP recording system. Phrenic nerve function was monitored during right PVI. Finally, a transthoracic echocardiogram excluded a pericardial effusion. A Femostop was applied. Two hundred and seventy-six patients were matched. Average age was 61 ± 0.7 years, 39% female. CHA2DS2 VASc score varied from 0 to 7. Procedure time was significantly shorter at the local hospital (63.5 ± 1.1 vs. 101.7 ± 2.9 min, P < 0.0001). Fluoroscopy time (5.5 ± 0.2 vs. 12.6 ± 0.6 min, P < 0.0001) and fluoroscopy dose were lower (17.2 ± 2.1 vs. 97.6 ± 14.6 mGy, P < 0.0001). Successful PVI was achieved in all. The complication rate was low (5.4% vs. 6.3%, P = not significant). Four (1.4%) patients were not day case discharges. At 3 month follow-up, 54.3% had complete resolution, and 26.1% had improvement of symptoms. A total of 16.6% patients requested repeat procedures for ongoing symptoms. CONCLUSION: In experienced hands, cryoballoon for paroxysmal AF is delivered safely and effectively in a local centre. Outcomes remain excellent. These short, day case procedures allow utilization of non-cardiac centres. The service provides a model to meet increasing demands.

Circulating serum-derived microparticles provide novel proteomic biomarkers of spontaneous preterm birth.

OBJECTIVE: The purpose of this study was to determine whether the proteomic biosignature of circulating microparticles in maternal serum obtained in the second trimester could identify pregnancies that result in spontaneous preterm birth (SPTB). STUDY DESIGN: Microparticles were isolated from blinded biorepository-sourced serum samples from 48 pregnant women at 15 to 17 weeks of gestation. Microparticle proteins were extracted and analyzed using label-free liquid chromatography/mass spectrometry. Peptide features were analyzed to assess the association of specific protein patterns with subjects delivering at term (≥ 37 weeks gestation; n = 24) and those experiencing SPTB (≤ 34 weeks gestation; n = 24). RESULTS: We found 99 proteins that had statistically significant differences in signal intensity between term and SPTB women in both first (n = 26) and second (n = 22) singleton gestation pregnancy cohorts. Additional evaluation identified 18 biomarkers that met criteria for further priority evaluation (12 preterm, 6 term). Pathway analysis showed that differentiating SPTB biomarker proteins were predominantly associated with inflammation and cell injury, while differentiating term biomarkers were associated with cell growth and hematological parameters. CONCLUSION: This study shows for the first time that the proteomic content of serum microparticles isolated in the second trimester can identify with a high degree of accuracy pregnancies that result in SPTB.

Fluid pressure is a magnitude-dependent modulator of early endothelial tubulogenic activity: implications related to a potential tissue-engineering control parameter.

A significant barrier to the success of engineered tissues is the inadequate transport of nutrients and gases to, and waste away from, cells within the constructs, after implantation. Generation of microtubular networks by endothelial cells in engineered constructs to mimic the in vivo transport scheme is essential for facilitating tissue survival by promoting the in vitro formation of microvessels that integrate with host microvasculature, after implantation. Previously, we reported that select pressures stimulate endothelial proliferation involving protubulogenic molecules such as fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor-C (VEGF-C). Based on this, we investigated fluid pressure as a selective modulator of early tubulogenic activity with the intent of assessing the potential utility of this mechanical stimulus as a tissue-engineering control parameter. For this purpose, we used a custom pressure system to expose two-dimensional (2D) and three-dimensional (3D) cultures of endothelial cells to static pressures of 0 (controls), 20, or 40 mmHg for 3 days. Compared to controls, 2D endothelial cultures exposed to 20, but not 40 mmHg, exhibited significantly (p<0.05) enhanced cell growth that depended on VEGF receptor-3 (VEGFR-3), a receptor for VEGF-C. Moreover, endothelial cells grown on microbeads and suspended in 3D collagen gels under 20 mmHg, but not 40 mmHg, displayed significantly (p<0.05) increased sprout formation. Interestingly, pressure-dependent proliferation and sprout formation occurred in parallel with pressure-sensitive upregulation of VEGF-C and VEGFR-3 expression and were sensitive to local FGF-2 levels. Collectively, the results of the present study provided evidence that early endothelial-related tubulogenic activity depends on local hydrostatic pressure levels in the context of local growth factor conditions. In addition to relevance to microvascular diseases associated with interstitial hypertension (e.g., cancer and glaucoma), these findings provided first insight into the potential utility of hydrostatic pressure as a fine-tune control parameter to optimize microvascularization of tissue-engineering constructs in the in vitro setting before their implantation.

Facilitated diffusion of VEGF165 through descemet's membrane with sucrose octasulfate.

Vascular endothelial growth factor A (VEGF-A) is a promoter of neovascularization and thus a popular therapeutic target for diseases involving excessive growth of blood vessels. In this study, we explored the potential of the disaccharide sucrose octasulfate (SOS) to alter VEGF165 diffusion through Descemet's membrane. Descemet's membranes were isolated from bovine eyes and used as a barrier between two chambers of a diffusion apparatus to measure VEGF transport. Diffusion studies revealed a dramatic increase in VEGF165 transport in the presence of SOS, with little diffusion of VEGF165 across the membrane over a 10-h time course in the absence of SOS. Diffusion studies with VEGF121, a non-heparin binding variant of VEGF, showed robust diffusion with or without SOS. To determine a possible mechanism, we measured the ability of SOS to inhibit VEGF interactions with extracellular matrix (ECM), using cell-free and cell surface binding assays. Binding studies showed SOS had no effect on VEGF165 binding to either heparin-coated plates or endothelial cell surfaces at less than mg/ml concentrations. In contrast, we show that SOS inhibited VEGF165 binding to fibronectin in a dose dependent manner and dramatically accelerated the rate of release of VEGF165 from fibronectin. SOS also inhibited the binding of VEGF165 to fibronectin-rich ECM deposited by vascular smooth muscle cells. These results suggest that fibronectin-rich extracellular matrices serve as barriers to VEGF165 diffusion by providing a network of binding sites that can trap and sequester the protein. Since the content of Descemet's membrane is typical of many basement membranes it is possible that they serve throughout the body as formidable barriers to VEGF165 diffusion and tightly regulate its bioavailability and distribution within tissues.

Endothelial cell capture of heparin-binding growth factors under flow.

Circulation is an important delivery method for both natural and synthetic molecules, but microenvironment interactions, regulated by endothelial cells and critical to the molecule's fate, are difficult to interpret using traditional approaches. In this work, we analyzed and predicted growth factor capture under flow using computer modeling and a three-dimensional experimental approach that includes pertinent circulation characteristics such as pulsatile flow, competing binding interactions, and limited bioavailability. An understanding of the controlling features of this process was desired. The experimental module consisted of a bioreactor with synthetic endothelial-lined hollow fibers under flow. The physical design of the system was incorporated into the model parameters. The heparin-binding growth factor fibroblast growth factor-2 (FGF-2) was used for both the experiments and simulations. Our computational model was composed of three parts: (1) media flow equations, (2) mass transport equations and (3) cell surface reaction equations. The model is based on the flow and reactions within a single hollow fiber and was scaled linearly by the total number of fibers for comparison with experimental results. Our model predicted, and experiments confirmed, that removal of heparan sulfate (HS) from the system would result in a dramatic loss of binding by heparin-binding proteins, but not by proteins that do not bind heparin. The model further predicted a significant loss of bound protein at flow rates only slightly higher than average capillary flow rates, corroborated experimentally, suggesting that the probability of capture in a single pass at high flow rates is extremely low. Several other key parameters were investigated with the coupling between receptors and proteoglycans shown to have a critical impact on successful capture. The combined system offers opportunities to examine circulation capture in a straightforward quantitative manner that should prove advantageous for biologicals or drug delivery investigations.

Vitamin D binding protein-macrophage activating factor directly inhibits proliferation, migration, and uPAR expression of prostate cancer cells.

BACKGROUND: Vitamin D binding protein-macrophage activating factor (DBP-maf) is a potent inhibitor of tumor growth. Its activity, however, has been attributed to indirect mechanisms such as boosting the immune response by activating macrophages and inhibiting the blood vessel growth necessary for the growth of tumors. METHODS AND FINDINGS: In this study we show for the first time that DBP-maf exhibits a direct and potent effect on prostate tumor cells in the absence of macrophages. DBP-maf demonstrated inhibitory activity in proliferation studies of both LNCaP and PC3 prostate cancer cell lines as well as metastatic clones of these cells. Flow cytometry studies with annexin V and propidium iodide showed that this inhibitory activity is not due to apoptosis or cell death. DBP-maf also had the ability to inhibit migration of prostate cancer cells in vitro. Finally, DBP-maf was shown to cause a reduction in urokinase plasminogen activator receptor (uPAR) expression in prostate tumor cells. There is evidence that activation of this receptor correlates with tumor metastasis. CONCLUSIONS: These studies show strong inhibitory activity of DBP-maf on prostate tumor cells independent of its macrophage activation.

A computational model of FGF-2 binding and HSPG regulation under flow.

A novel convection-diffusion-reaction model is developed to simulate fibroblast growth factor (FGF-2) binding to cell surface receptors (FGFRs) and heparan sulfate proteoglycans (HSPGs) under flow conditions within a cylindrical-shaped vessel or capillary. The model consists of a set of coupled nonlinear partial differential equations (PDEs) and a set of coupled nonlinear ordinary differential equations (ODEs). The time-dependent PDE system is discretized and solved by a second-order implicit Euler scheme using the finite volume method. The ODE system is solved by a stiff ODE solver VODE using backward differencing formulation (BDF). The transient solution of FGF-2, FGFR, HSPG, and their bound complexes for three different flow rates are computed and presented. Simulation results indicate that the model can predict growth factor transport and binding to receptors with/without the presence of heparan sulfate, as well as the effect of flow rate on growth factor-receptor binding. Our computational model may provide a useful means to investigate the impact of fluid flow on growth factor dynamics, and ultimately, signaling within the circulation.

Control of growth factor networks by heparan sulfate proteoglycans.

Growth factor binding to transmembrane protein receptors is generally understood to initiate cell signaling. Receptor binding of heparin-binding growth factors (HB-GFs), such as fibroblast growth factor-2 (FGF-2), is regulated by interactions with heparan sulfate proteoglycans. While there is some specificity for binding to heparan sulfate, overlap in sites for different growth factors may allow for cross regulation. Here we demonstrate, using experiments and computer simulations, that the HB-GFs FGF-2 and heparin-binding EGF-like growth factor (HB-EGF) can cross regulate receptor binding of the other despite having unique receptors. The ability of HSPG to stabilize HB-GF receptor binding is critical for competing growth factors to modulate receptor binding with both enhanced and reduced binding possible depending on this stabilization process. HSPG density and affinity for HB-GF are also critical factors for HB-GF cross regulation. Simulations further reveal that HB-GF can regulate receptor binding of non-HB-GFs such as EGF even when the two proteins share no binding sites when other HB-GF are present within the network. Proliferation studies demonstrate potentiation of HB-EGF-induced growth by FGF-2 indicating that competition networks can alter biological response. Exogenous manipulation of cellular responses to growth factors in complex living systems will require understanding the HSPG-controlled network.

Sucrose octasulfate regulates fibroblast growth factor-2 binding, transport, and activity: potential for regulation of tumor growth.

The antithrombotic activity of heparin has largely been credited with the success found in some cancer treatment by heparin. There are, however, many potent growth factors involved in tumor and blood vessel growth that bind to heparin with high affinity and their regulation by heparin may play a role in heparin's efficacy. We therefore chose to study the activity of a heparin analog, sucrose octasulfate (SOS), which has been similarly shown to interact with heparin-binding growth factors. Using mouse melanoma and lung carcinoma models, we demonstrate in vivo inhibition of tumor growth by SOS. SOS, however, showed little effect in coagulation assays indicating that this activity was not a primary mechanism of action for this molecule. Studies were then performed to assess the effect of SOS on basic fibroblast growth factor (FGF-2) activity, a growth factor which promotes tumor and blood vessel growth and is produced by B16 melanoma cells. SOS potently inhibited FGF-2 binding to endothelial cells and stripped pre-bound FGF-2 from cells. SOS also regulated FGF-2 stimulated proliferation. Further, SOS facilitated FGF-2 diffusion through Descemet's membrane, a heparan sulfate-rich basement membrane from the cornea, suggesting a possible role in FGF-2 clearance. Our results suggest that molecules such as SOS have the potential to remove growth factors from tumor microenvironments and the approach offers an attractive area for further study.

PPARalpha agonist fenofibrate suppresses tumor growth through direct and indirect angiogenesis inhibition.

Angiogenesis and inflammation are central processes through which the tumor microenvironment influences tumor growth. We have demonstrated recently that peroxisome proliferator-activated receptor (PPAR)alpha deficiency in the host leads to overt inflammation that suppresses angiogenesis via excess production of thrombospondin (TSP)-1 and prevents tumor growth. Hence, we speculated that pharmacologic activation of PPARalpha would promote tumor growth. Surprisingly, the PPARalpha agonist fenofibrate potently suppressed primary tumor growth in mice. This effect was not mediated by cancer-cell-autonomous antiproliferative mechanisms but by the inhibition of angiogenesis and inflammation in the host tissue. Although PPARalpha-deficient tumors were still susceptible to fenofibrate, absence of PPARalpha in the host animal abrogated the potent antitumor effect of fenofibrate. In addition, fenofibrate suppressed endothelial cell proliferation and VEGF production, increased TSP-1 and endostatin, and inhibited corneal neovascularization. Thus, both genetic abrogation of PPARalpha as well as its activation by ligands cause tumor suppression via overlapping antiangiogenic pathways. These findings reveal the potential utility of the well tolerated PPARalpha agonists beyond their use as lipid-lowering drugs in anticancer therapy. Our results provide a mechanistic rationale for evaluating the clinical benefits of PPARalpha agonists in cancer treatment, alone and in combination with other therapies.

Dose-dependent response of FGF-2 for lymphangiogenesis.

Spatio-temporal studies on the growth of capillary blood vessels and capillary lymphatic vessels in tissue remodeling have suggested that lymphangiogenesis is angiogenesis-dependent. We revisited this concept by using fibroblast growth factor 2 (FGF-2) (80 ng) to stimulate the growth of both vessel types in the mouse cornea. When we lowered the dose of FGF-2 in the cornea 6.4-fold (12.5 ng), the primary response was lymphangiogenic. Further investigation revealed that vascular endothelial growth factor-C and -D are required for this apparent lymphangiogenic property of FGF-2, and when the small amount of accompanying angiogenesis was completely suppressed, lymphangiogenesis remained unaffected. Our findings demonstrate that there is a dose-dependent response of FGF-2 for lymphangiogenesis, and lymphangiogenesis can occur in the absence of a preexisting or developing vascular bed, i.e., in the absence of angiogenesis, in the mouse cornea.

Binding inhibition of angiogenic factors by heparan sulfate proteoglycans in aqueous humor: potential mechanism for maintenance of an avascular environment.

Aqueous humor is a clear fluid, primarily a blood filtrate, which circulates through the anterior chamber of the eye and bathes the cornea. We explored the possibility that components in the aqueous humor play a direct part in maintaining the avascular environment of the cornea. We report here that heparan sulfate proteoglycan (HSPG) was found in bovine aqueous humor and that it directly inhibits binding of basic fibroblast growth factor and vascular endothelial growth factor to cell-surface heparan sulfate. We demonstrate that this holds true for all heparin binding proteins tested but not for epidermal growth factor, which does not bind heparin. Furthermore, we show, with mathematical modeling, that the concentration of HSPG in aqueous humor (approximately 4 microg/ml), when combined with the clearance of aqueous humor from the eye due to circulation, is sufficient to block the binding of heparin binding growth factors to corneal endothelium. This mechanism suggests a physiological process to control bioavailability of angiogenic growth factors in the cornea.

Vitamin D binding protein-macrophage activating factor (DBP-maf) inhibits angiogenesis and tumor growth in mice.

We have isolated a selectively deglycosylated form of vitamin D binding protein (DBP-maf) generated from systemically available DBP by a human pancreatic cancer cell line. DBP-maf is antiproliferative for endothelial cells and antiangiogenic in the chorioallantoic membrane assay. DBP-maf administered daily was able to potently inhibit the growth of human pancreatic cancer in immune compromised mice (T/C=0.09). At higher doses, DBP-maf caused tumor regression. Histological examination revealed that treated tumors had a higher number of infiltrating macrophages as well as reduced microvessel density, and increased levels of apoptosis relative to untreated tumors. Taken together, these data suggest that DBP-maf is an antiangiogenic molecule that can act directly on endothelium as well as stimulate macrophages to attack both the endothelial and tumor cell compartment of a growing malignancy.