Von Willebrand Factor Staining Research Articles

FGF2 is crucial for the development of bovine luteal endothelial networks in vitro

The development of the corpus luteum requires angiogenesis, and involves the complex interplay between factors such as vascular endothelial growth factor A (VEGFA), fibroblast growth factor 2 (FGF2) and platelet-derived growth factor (PDGF). However, the relative role of these factors remains to be elucidated. This study used a new physiologically relevant mixed luteal cell culture system to test the hypotheses that: a) FGF2 and VEGFA are critical for bovine luteal angiogenesis; and b) local luteal PDGF signalling stimulates the formation of endothelial networks. Cells were treated with receptor tyrosine kinase inhibitors against VEGFA (SU1498), FGF2 (SU5402) or PDGF (AG1295) activity. After 9 days in culture, endothelial cells were immunostained for von Willebrand factor (VWF) and quantified by image analysis. Highly organised intricate endothelial networks were formed in the presence of exogenous VEGFA and FGF2. The inhibition of FGF2 activity reduced the total area of VWF staining versus controls (>95%; P<0.001). Inhibition of VEGF and PDGF activity reduced the endothelial network formation by more than 60 and 75% respectively (P<0.05). Progesterone production increased in all treatments from day 1 to 7 (P<0.001), and was unaffected by FGF2 or PDGF receptor kinase inhibition (P>0.05), but was reduced by the VEGF receptor inhibitor on days 5 and 7 (P<0.001). In conclusion, this study confirmed that VEGF signalling regulates both bovine luteal angiogenesis and progesterone production. However, FGF2 was crucial for luteal endothelial network formation. Also, for the first time, this study showed that local luteal PDGF activity regulates bovine luteal endothelial network formation in vitro.

Induced Endothelial Cells Enhance Osteogenesis and Vascularization of Mesenchymal Stem Cells

Adequate vascularization remains one of the major challenges in bone tissue engineering. Since the microvascular endothelium is of benefit to osteogenesis and vascularization when in direct contact with bone marrow mesenchymal stem cells (BM-MSCs), we investigated whether endothelial cells induced from BM-MSCs have the same effect on BM-MSCs in vitro and in vivo. BM-MSCs were isolated, characterized and induced into endothelial-like cells (induced endothelial cells, IECs) in endothelial cell growth medium 2. BM-MSCs and IECs were co-cultured with direct contact. In vitro, IECs were evaluated in terms of their characteristics of endothelial cells and their effects on the osteogenic potential of BM-MSCs by cell morphology, immunofluorescent staining, alkaline phosphatase activity and osteocalcin synthesis. In vivo, scaffolds consisting of β-tricalcium phosphate co-seeded with IECs and BM-MSCs were transplanted into mouse dorsal pockets, and a histological analysis was performed to determine the extent of new bone and blood vessel formation. Isolated BM-MSCs were positive for the markers CD105 and CD29 and negative for hematopoietic markers CD34, CD45 and CD14. They were able to differentiate into adipocytes, osteocytes and chondrocytes in respective media. Immunofluorescent analysis with von Willebrand factor and CD31 staining showed that BM-MSCs could differentiate into endothelial cells. The alkaline phosphatase activity and the osteocalcin content of the co-culture group were obviously higher than those of any other group (p < 0.05). Histologically, newly formed bone and vessels were more evident in the culture group (p < 0.05). Our findings suggest that IECs could efficiently stimulate the in vitro differentiation of osteoblast-like cells and promote osteogenesis in vivo by direct contact with BM-MSCs.

Immunohistochemical Examination of Complex Vascular Lesions in the Lungs of Domestic Fowl Selected for Susceptibility to Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a human disease in which vascular remodeling can lead to the formation of plexiform lesions in terminal pulmonary arterioles. Various molecules are expressed by the diverse cell types within human plexiform lesions, including smooth muscle actin (SMA), von Willebrand factor (VWF), vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), collagen, and immune cells such as macrophages and CD8+ cells. Plexiform lesions rarely develop in the lungs of the laboratory mammals, but plexiform‐like complex vascular lesions (CVL) do develop spontaneously in the lungs of chickens from a PAH‐susceptible line. This study examined the expression of various molecules by the cells within CVL in chicken lungs. Paraformaldehyde fixed, paraffin embedded sections of lungs from 8‐ to 24‐week old male and female chickens were stained immunohistochemically using monoclonal or polyclonal antibodies. Cells within chicken CVL exhibited positive staining for SMA, VWF, VEGF, VEGFR2, collagen type III, macrophage and CD8 molecules. Foam‐like giant cells within the CVL stained positively as macrophages, whereas positive staining for VWF was detected in tufts of intimal cells and in the capillary endothelium of CVL. Immunohistochemical staining patterns within the CVL of PAH‐susceptible chickens combined with the close histological resemblance of CVL to plexiform lesions of human PAH patients further validates chickens from our PAH‐susceptible line as an excellent animal model of spontaneous plexogenic arteriopathy. Supported by NIH grant 1R15HL092517.

A method for identification of vocal fold lamina propria fibroblasts in culture

Vocal fold biology research is emerging as a vital area of study in laryngology. One impediment is the lack of both commercially available vocal fold lamina propria fibroblasts and a constitutively expressed specific marker for fibroblasts. We present an in vitro technique that allows for identification of fibroblasts by ruling out the possibility of the cells belonging to other lineages that are found in vocal fold tissue. An in vitro study. Two primary vocal fold fibroblast cell lines and one immortalized vocal fold fibroblast cell line were cultured. Immunohistologic staining for alpha-actinin, cytokeratin 19, and von Willebrand factor was completed for the three fibroblast lines in addition to skeletal, endothelial, and epithelial cell lines. Cell type was differentiated by positive staining for alpha-actinin, cytokeratin 19, and von Willebrand factor. Fibroblast cultures did not express alpha-actinin, cytokeratin 19, and von Willebrand factor, whereas skeletal muscle, endothelial, and epithelial cultured cells expressed each respectively. This simple rule-out methodology for fibroblast confirmation is an important step when establishing cell culture, and it establishes sound internal validity particularly in the early stages of this emerging area of study.

Induction of Pulmonary Thromboembolism by Neutrophil Elastase in Collagen-Induced Arthritis Mice and Effect of Recombinant Human Soluble Thrombomodulin

We previously reported that during total knee arthroplasty in rheumatoid arthritis (RA) patients, the use of tourniquet might promote local release of neutrophil elastase (NE) from neutrophils, which may contribute to the development of pulmonary thromboembolism (PTE) and tissue injury. The aim of this study was to develop PTE by the use of NE in a mouse model of collagen-induced arthritis (CIA) and investigate the relationship between thrombus and endothelial cells as well as the effect of recombinant human soluble thrombomodulin (rhs-TM) in reducing the risk of PTE. Male DBA/1J mice were injected intracutaneously at several sites with an emulsion containing bovine collagen and later a booster shot to produce CIA mice. Subsequently, NE was injected intravenously 2 times a day for 3 days and after a further 4 days, mice were sacrificed. A group of mice received rhs-TM injections prior to NE injections. We divided the mice into four groups of normal, CIA control, CIA + NE, and CIA + rhs-TM + NE mice and evaluated thrombus formation status. All CIA + NE mice developed PTE. In contrast, no thrombosis was found in normal control, CIA control and CIA + rhs-TM + NE mice. Plasma thrombin level, fibrinogen expression and neutrophil count were increased in CIA + NE mice. Double staining for anticoagulant TM and procoagulant von Willebrand factor (vWF) in pulmonary endothelial cells in normal mice showed a TM-dominant expression while in both CIA control and CIA + NE mice a vWF-dominant expression compatible with coagulant status was observed. Injection of rhs-TM into CIA + NE mice resulted in a phenotypic conversion of endothelial cells from vWF-dominant to TM-dominant expression and a reduction in fibrinogen deposition. These findings demonstrate that by repeated use of NE in CIA mice, it is feasible to produce PTE and to study its pathogenesis and that rhs-TM reduces the risk of PTE. We suggest that in surgical operations of upper and lower extremities in RA patients, the use of a tourniquet should be avoided as it may trigger NE release.

Bioengineered Three-Layered Robust and Elastic Artery Using Hemodynamically-Equivalent Pulsatile Bioreactor

There is an essential demand for tissue engineered autologous small-diameter vascular graft, which can function in arterial high pressure and flow circulation. We investigated the potential to engineer a three-layered robust and elastic artery using a novel hemodynamically-equivalent pulsatile bioreactor. Endothelial cells (ECs), smooth muscle cells (SMCs), and fibroblasts were harvested from bovine aorta. A polyglycolic acid (PGA) sheet and a polycaprolactone sheet seeded with SMCs, and a PGA sheet seeded with fibroblast, were wrapped in turn on a 6-mm diameter silicone tube and incubated in culture medium for 30 days. The supporting tube was removed, and the lumen was seeded with ECs and incubated for another 2 days. The pulsatile bioreactor culture, under regulated gradual increase in flow and pressure from 0.2 (0.5/0) L/min and 20 (40/15) mm Hg to 0.6 (1.4/0.2) L/min and 100 (120/80) mm Hg, was performed for an additional 2 weeks (n=10). The engineered vessels acquired distinctly similar appearance and elasticity as native arteries. Scanning electron microscopic examination and Von Willebrand factor staining demonstrated the presence of ECs spread over the lumen. Elastica Van Gieson and Masson Tricrome Stain revealed ample production of elastin and collagen in the engineered grafts. Alpha-SMA and calponin staining showed the presence of SMCs. Tensile tests demonstrated that engineered vessels acquired equivalent ultimate strength and similar elastic characteristics as native arteries (Ultimate Strength of Native: 882+/-133 kPa, Engineered: 827+/-155 kPa, each n=8). A robust and elastic small-diameter artery was engineered from three types of vascular cells using the physiological pulsatile bioreactor.

Combined Bcl 2 /mTOR Inhibition Leads to Enhanced Radiosensitization via Induction of Autophagy and Apoptosis in Non-small Lung Tumor Xenograft Model

Radiotherapy has a central role in the treatment of locally advanced non-small cell lung cancer. The effectiveness of this modality, however, is often limited, in part due to the presence of defects in cell death pathways. In this study, we tested whether co-upregulation of autophagy and apoptosis can be utilized to enhance radiation sensitivity of lung cancer cells in vitro and in a lung cancer xenograft model. Clonogenic assay was used to determine radiosensitivity. H460 lung cancer xenografts was generated and immunohistochemistry (IHC) was performed on tumor sections for tumor cell proliferation (Ki67 staining), angiogenesis (von Willebrand factor staining), and apoptosis (cleaved caspase-3 antibody staining). Autophagy was determined by punctate localization of GFP-LC3 fusion protein, which observed under a confocal fluorescence microscope in vitro. Apoptosis measured using the Annexin V-fluorescent isothiocyante apoptosis detection kit I (Pharmingen) with flow cytometry in vitro. Autophagy was induced by rapamycin, an mTOR inhibitor, and apoptosis was induced by AT-737, a Bcl2 inhibitor. The combination of AT-737 and rapamycin markedly enhanced sensitivity of H460 cells to radiation (DER = 2.28, p = 0.001) in clonogenic assay. This enhanced radiosensitization was associated with an increase in characteristic punctate localization of GFP-LC3 and in Annexin-V positive cells, suggesting that both apoptosis and autophagy were induced simultaneously. In addition, the concurrent administration of AT-737, rapamycin and radiation in vivo significantly decreased cell proliferation by 77% (p < 0.001) and vascular density by 67.5% (p < 0.008) compared to radiation alone. Consistent with the in vitro results, the tri-modality treatment showed a significant 14-fold increase in caspase-3 activity as a marker for apoptosis in the lung tumor xenograft model. These results suggest that concurrent upregulation of autophagy and apoptosis may be utilized as a novel strategy to enhance radiation therapy in lung cancer.

VEGF Gene Therapy Augments Localized Angiogenesis and Promotes Anastomotic Wound Healing: A Pilot Study in a Clinically Relevant Animal Model

BackgroundAnastomotic leak related to ischemia is a source of significant morbidity and mortality in gastrointestinal surgery. The aim of this study was to apply growth factor gene transfection for the purpose of up-regulating angiogenesis, increasing anastomotic strength, and ultimately preventing dehiscence. MethodsAn opossum esophagogastrostomy model was employed. The human vascular endothelial growth factor (VEGF165) gene was incorporated into a recombinant plasmid. The VEGF plasmid vector was then complexed with a cationic synthetic carrier, polyethyleneimine. Control animals received plasmid devoid of VEGF165 (n = 6). The experimental group received VEGF165 plasmid (n = 5). After esophagogastrectomy and gastric tubularization, plasmid was injected into the submucosa of the neoesophagus at the anastomotic site. Conduit arteriography was performed before and 10 days after injection. Euthanasia occurred on post-injection day 10 and the anastomosis was removed en bloc. A second group of animals treated with VEGF165 were euthanized 30 and 37 days post injection. Blood flow was measured with laser-Doppler prior to euthanasia. Ex vivo anastomotic bursting pressure was performed. Tissue samples were procured for RNA extraction and von Willebrand Factor staining. Microvessel counts were obtained by two blinded observers. Tissue VEGF transcript levels were measured with reverse transcriptase polymerase chain reaction (RT-PCR). ResultsThere was one anastomotic leak in the control group. Experimental animals demonstrated significantly increased bursting pressure (104.25±6.2 vs 86.73±9.4 mmHg, p = 0.021) and neovascularization (33.87±9.6 vs 20.33±8.1 vessels/hpf, p = 0.032) compared to controls. In addition, there was a strongly positive correlation between the number of microvessels and bursting pressure (r = 0.808, p = 0.015, Pearson’s). On angiographic examination, treated animals demonstrated more neovascularization compared to controls. RT-PCR demonstrated up to a 5.6-fold increase in VEGF mRNA in treated compared to controls. DiscussionThis description of gene therapy in gastrointestinal surgery using VEGF165 transfection demonstrates increased angiogenesis with subsequently improved anastomotic healing in a clinically relevant model.

Progesterone and Placentation Increase Secreted Phosphoprotein One (SPP1 or Osteopontin) in Uterine Glands and Stroma for Histotrophic and Hematotrophic Support of Ovine Pregnancy1

Secreted phosphoprotein one (SPP1, osteopontin) may regulate conceptus implantation and placentation. We investigated effects of progesterone (P(4)) and the conceptus on expression and localization of SPP1 in the ovine uterus. Steady-state levels of SPP1 mRNA in the endometrium of unilaterally pregnant ewes did not differ significantly between nongravid and gravid horns within their respective days of pregnancy; however, levels did increase as pregnancy progressed. SPP1 mRNA was detectable in the glandular epithelium (GE) of both nongravid and gravid horns via in situ hybridization. SPP1 protein was localized to the apical surface of the luminal epithelium of both nongravid and gravid uterine horns. Gravid horns exhibited extensive stromal SPP1 on Days 40 through 120, whereas SPP1 was markedly lower in the stroma of nongravid uterine horns through Day 80 of pregnancy. By Day 120, stromal expression of SPP1 between nongravid and gravid horns was similar. Long-term P(4) treatment of ovariectomized ewes induced SPP1 in the uterine stroma and GE. A bioactive 45-kDa SPP1 fragment was purified from uterine secretions and promoted ovine trophectoderm cell attachment in vitro. Interestingly, increased stromal cell expression of SPP1 was positively associated with vascularization as assessed by von Willebrand factor staining. Finally, ovine uterine artery endothelial cells produced SPP1 during outgrowth into three-dimensional collagen matrices in an in vitro model system that recapitulates angiogenesis. Collectively, P(4) induces and the conceptus further stimulates SPP1 in uterine GE and stroma, where SPP1 likely influences histotrophic and hematotrophic support of conceptus development.

069 The Role of Papain-Urea-Copper Chlorophyllin Active Components Tested Single-Variably in Acute Infected Pig Wounds

One of the well-known enzymatic debriders is a papain–urea-chlorophyllin ointment. The purpose of this study was to determine the efficacy of this ointment compared with single variable Papain-urea, Copper chlorophyllin, or Urea base ointment and moist wound care in porcine full thickness wounds. Twenty full thickness wounds were made on the dorsum of domestic pigs. Following hemostasis, the wounds were contaminated with wound-isolated bacteria, treated with test articles, and dressed with moisture-retentive dressing. All the wounds were evaluated 1, 4, 8, 11, 14, 18, and 21 days postsurgery visually and biopsies were obtained for microscopy. The results showed no impairment in the rate of epithelialization of the papain–urea-chlorophyllin treatment, nor with single variable actives compared with moist wound care. Microscopic examination revealed an increase in the number of keratinocytes present in the epidermis of the papain–urea-chlorophyllin and papain-treated wounds, and an increase in the depth of rete pegs. Blood vessel formation was increased in the papain–urea-chlorophyllin, papain, and Copper chlorophyllin groups compared to the moist control tissue as seen with CD31 and von Willebrand Factor staining, and was most evident in the papain–urea-chlorophyllin-treated tissues. VEGF was increased after 1 week in the wounds treated with papain–urea-chlorophyllin and papain compared to vehicle, indicating a possible mechanism for neovascularization. The increase was not seen in the Copper chlorophyllin group, indicating the possibility of another mechanism for blood vessel formation in this group. The healing appears to be most complete in the papain–urea-chlorophyllin group based on epidermal morphology and enhanced vascularization, which is vital in supplying nutrients to the newly healed wound.

080 The Effects of Papain-Urea-Copper Chlorophyllin Ointment on Healing of Wounds in an Acute Infected Full Thickness Porcine Model

Removal of necrotic tissue is essential for wound-bed preparation for the treatment of chronic wounds. Enzymatic debridement appears to be most useful in the removal of eschar when surgical techniques cannot be utilized. The purpose of this study is to determine the efficacy of papain–urea chlorophyllin compared to moist wound care using an infected, acute, porcine full thickness wound model. Twenty full thickness wounds were created on the dorsum of domestic pigs. Following hemostasis, the wounds were contaminated with wound-isolated bacteria then treated with test articles and dressed with moisture-retentive dressing. All the wounds were evaluated 1, 4, 8, 11, 14,18, and 21 days postsurgery for epithelialization and biopsies were obtained for microscopic evaluation. The results showed no adverse effect in reepithelialization in acute wounds when using papain–urea chlorophyllin ointment compared to moist wound care, TEWL measurements also showed no adverse affects from the treatment. Microscopic examination reveals an increase in the number of keratinocytes present in the epidermis of the treated pigs as well as an increase in the depth of rete pegs. New blood vessel formation was increased at all examined time points in the papain–urea chlorophyllin treated pigs, which was verified with CD31 and von Willebrand Factor staining. VEGF was increased after 1 week in the wound fluid from treated wounds compared to moist control. While the reepithelialization rate does not appear to be inhibited by papain–urea-chlorophyllin combination in acute wounds, the healing appears to be more complete based on the number of keratinocytes present in the epidermis and the extensive rete peg formation. The wounds also appear to have enhanced vascularization which is vital in supplying nutrients to the newly healed wound.

M867, a Novel Selective Inhibitor of Caspase-3 Enhances Cell Death and Extends Tumor Growth Delay in Irradiated Lung Cancer Models

BackgroundLung cancer remains the leading cause of cancer death worldwide. Radioresistance of lung cancer cells results in unacceptable rate of loco-regional failure. Although radiation is known to induce apoptosis, our recent study showed that knockdown of pro-apoptotic proteins Bak and Bax resulted in an increase in autophagic cell death and lung cancer radiosensitivity in vitro. To further explore the potential of apoptosis inhibition as a way to sensitize lung cancer for therapy, we tested M867, a novel chemical and reversible caspase-3 inhibitor, in combination with ionizing radiation in vivo and in vitro.Methods and FindingsM867 reduced clonogenic survival in H460 lung cancer cells (DER = 1.27, p = 0.007) compared to the vehicle-treated treated cells. We found that administration of M867 with ionizing radiation in an in vivo mouse hind limb lung cancer model was well tolerated, and produced a significant tumor growth delay compared to radiation alone. A dramatic decrease in tumor vasculature was observed with M867 and radiation using von Willebrand factor staining. In addition, Ki67 index showed >5-fold reduction of tumor proliferation in the combination therapy group, despite the reduced levels of apoptosis observed with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining. Radiosensitizing effect of M867 through inhibiting caspases was validated using caspase-3/-7 double-knockout (DKO) mouse embryonic fibroblasts (MEF) cell model. Consistent with our previous study, autophagy contributed to the mechanism of increased cell death, following inhibition of apoptosis. In addition, matrigel assay showed a decrease in in vitro endothelial tubule formation during the M867/radiation combination treatment.ConclusionsM867 enhances the cytotoxic effects of radiation on lung cancer and its vasculature both in vitro and in vivo. M867 has the potential to prolong tumor growth delay by inhibiting tumor proliferation. Clinical trials are needed to determine the potential of this combination therapy in patients with locally advanced lung cancer.

Microvascular Injury, Thrombosis, Inflammation, and Apoptosis in the Pathogenesis of Heatstroke

Severe heatstroke is a leading cause of morbidity and mortality during heat waves. The pathogenesis of tissue injury, organ failure, and death in heatstroke is not well understood. We investigated the pathways of heatstroke-induced tissue injury and cell death in anesthetized baboons (Papio hamadyras) subjected to environmental heat stress until core temperature attained 42.5 degrees C (moderate heatstroke; n = 3) or onset of severe heatstroke (n = 4) signaled by a fall in systolic blood pressure to < 90 mm Hg and rise in core temperature to 43.1+/-0.1 degrees C. Three sham-heated animals served as controls. Light and electron microscopy revealed widespread hemorrhage and thrombosis, transmural migration of leukocytes, and microvascular endothelium injury in severe heatstroke. Immunohistology and ultrastructural analysis demonstrated increased staining of endothelial von Willebrand factor (vWF), tissue factor (TF), and endothelial leukocyte-platelet interaction. Extensive apoptosis was noted in spleen, gut, and lung, and in hematopoeitic cells populating these organs. Double-labeling studies colocalized active caspase-3 and TF with apoptotic cells. Findings in sham-heated animals were unremarkable. These data suggested that microvascular injury, thrombosis, inflammation, and apoptosis may play an important role in the pathogenesis of heatstroke injury.

Distribution of Lymphatic Vessel Network in Normal Urinary Bladder

The lymphatic vessel endothelial hyaluronan receptor (LYVE-1) is a novel lymphatic vessel marker that is expressed on lymph vessel endothelial cells. The objective of this study was to determine the LYVE-1 expression patterns in normal urothelium and to compose the geometric topography of the lymphatic network. Immunohistochemical staining for LYVE-1 and von Willebrand factor was performed to assess the differences in the distribution of lymphatic vessels between the components in the urinary bladder. The sizes of the individual lymphatic vessels were categorized as small, medium, and large. To compare the lymphatic density (counts per square millimeter), the number of lymphatic vessels of the five random areas was counted in each specimen. LYVE-1 expression and the lymphatic density of the muscularis propria were significantly greater than those of other layers, including the epithelium, lamina propria, perivesical fat, and serosa (P <0.0001). The size of the lymphatic vessels in the muscularis propria was significantly smaller than that in the other layers (P <0.0001). The greatest distribution of the lymphatic vessels was identified in the border areas consisting of the lamina propria and muscular propria or the muscular propria and adventitia, including the perivesical fat and serosa. We have developed a new type of color geometric topography to accurately exhibit the distribution of that complex network. Lymphatic vessels, although small, are predominately distributed in the muscularis propria in the normal urothelium compared with that in the other layers. The LYVE-1 antibody was a useful specific marker for the illustration of the lymphatic vessel in conventional paraffin-embedded specimens of the urinary bladder.

Vein wall re-endothelialization after deep vein thrombosis is improved with low-molecular-weight heparin

Vein wall endothelial turnover after stasis deep vein thrombosis (DVT) has not been well characterized. The purpose of this study was to quantify re-endothelialization after DVT and determine if low-molecular-weight heparin (LMWH) therapy affects this process. Stasis DVT was generated in the rat by inferior vena cava ligation, with harvest at 1, 4, and 14 days. Immunohistologic quantification of vascular smooth muscle cells and luminal endothelialization was estimated by positive staining for alpha-smooth muscle actin and von Willebrand factor, respectively. In separate experiments, rats were treated either before or after DVT with subcutaneous LMWH (3 mg/kg daily) until harvesting at 4 and 14 days. The inferior vena cava was processed for histologic analysis or was processed for organ culture after the thrombus was gently removed. The vein wall was stimulated in vitro with interleukin-1beta (1 ng/mL), and the supernatant was processed at 48 hours for nitric oxide. Cells were processed by real-time polymerase chain reaction for endothelial nitric oxide synthase, inducible nitric oxide synthase, cyclooxygenase-1 and -2, and thrombomodulin at 4 and 14 days, and collagen I and III at 14 days. Comparisons were done with analysis of variance or t test. A P < .05 was significant. Thrombus size peaked at 4 days, whereas luminal re-endothelialization increased over time (1 day, 11% +/- 2%; 4 days, 23% +/- 4%; 14 days, 64% +/- 7% (+) von Willebrand factor staining; P < .01, n = 3 to 4, compared with non-DVT control). Similarly, vascular smooth muscle cell staining was lowest at day 1 and gradually returned to baseline by 14 days. Both before and after DVT, LMWH significantly increased luminal re-endothelialization, without a difference in thrombus size at 4 days, but no significant difference was noted at 14 days despite smaller thrombi with LMWH treatment. Pretreatment with LMWH was associated with increased vascular smooth muscle cell area and recovery of certain inducible endothelial specific genes. No significant difference in nitric oxide levels in the supernatant was found at 4 days. At 14 days, type III collagen was significantly elevated with LMWH treatment. Venous re-endothelialization occurs progressively as the DVT resolves and can be accelerated with LMWH treatment, although this effect appears limited to the early time frame. These findings may have clinical relevance for LMWH timing and treatment compared with mechanical forms of therapy. How the vein wall endothelium responds after deep vein thrombosis (DVT) has not been well documented owing to limited human specimens. This report shows that low-molecular-weight heparin accelerates or protects the endothelium and preserves medial smooth muscle cell integrity after DVT, but that this effect is limited to a relatively early time period. Although most DVT prophylaxis is pharmacologic (a heparin agent), use of nonpharmacologic measures is also common. The use of heparin prophylaxis, compared with after DVT treatment, and the acceleration of post-DVT re-endothelialization require clinical correlation.

Endothelial cells of human colorectal cancer and healthy colon reveal phenotypic differences in culture

Antiangiogenic drugs have been used successfully for the treatment of colorectal cancer (CRC). Viable tumor endothelial cells (TEC) and normal endothelial cells (NEC) of uninvolved colon tissue of the same patient have not been available to optimize treatment strategies in vitro. Therefore, our target was to establish a protocol for the isolation of TEC and NEC. These cells were isolated with very high purity via magnetic cell sorting of tissue samples obtained from CRC and healthy colon of eight patients. TEC and NEC expressed CD31, CD105, VE-cadherin, VCAM-1, ICAM-1 and E-selectin, formed capillaries in basal membrane extract and were able to take up acetylated low-density lipoprotein. They were negative for podoplanin, CD45, CD68 and cytokeratin-20 indicating blood vessel endothelial lineage. Intense staining of von Willebrand factor (vWF) was observed in five of eight NEC cultures, whereas vWF was absent or only slightly expressed in all TEC cultures in vitro. Low intracellular concentration of vWF was also detected in TEC and NEC at the tissue level. This demonstrated that differences exhibited by TEC and NEC in vivo are stably perpetuated in culture. The isolated cultures may provide a useful in vitro model to elucidate epigenetic effects on angiogenesis in cancer and to optimize antiangiogenic therapy.

Rho‐kinase activation in endothelial cells contributes to expansion of infarction after focal cerebral ischemia

Microcirculatory disturbances contribute to the expansion of infarct lesions after focal cerebral ischemia. Recently, it was shown that Rho-kinase involves in endothelial dysfunction via down-regulation of endothelial nitric oxide synthase function in a rodent stroke model. However, it is not clear whether endothelial Rho-kinase is activated in vivo or Rho-kinase activation contributes to microcirculatory disturbances after cerebral ischemia. In this study, we assessed the temporal and spatial profiles of Rho-kianse activity and the effect of the Rho-kinase inhibitor fasudil on microcirculatory disturbances in the focal brain ischemia. Rho-kinase activation was evaluated by analyzing the phosphorylation of adducin, a substrate of Rho-kinase, by immunohistochemistry. Staining for p-adducin was found in endothelia in the ischemic area 6 hr after induction of ischemia. Microcirculatory disturbances and increased endothelial cell staining for von Willebrand factor (vWF) were observed in the same area. Postischemic treatment with fasudil suppressed endothelial Rho-kinase activation, preserved microcirculation, and inhibited endothelial cell vWF staining. These effects resulted in inhibition of infarct expansion and improvement of neurologic deficits. These findings indicate that Rho-kinase is activated in the endothelial cells and contributes to microcirculatory disturbances in cerebral ischemia. The vascular protective effect of Rho-kinase inhibitors may be useful in the treatment of the acute phase of ischemic stroke.

Evidence that differential packaging of the major platelet granule proteins von Willebrand factor and fibrinogen can support their differential release.

von Willebrand factor (VWF) and fibrinogen are major storage proteins of platelet alpha-granules. VWF is synthesized by the megakaryocyte, the cell from which platelets bud, and fibrinogen is delivered to alpha-granules by endocytosis. Considering biosynthetic origins, VWF and fibrinogen might be differentially packaged within platelets. We applied immunofluorescence microscopy to provide whole platelet, global information on the distributions of VWF and fibrinogen. The distribution of VWF and fibrinogen were characterized in both the resting state and handling activated human platelets. Full cell volume image stacks were collected by spinning-disk confocal microscopy, corrected for a small pixel shift between green and red channels, deconvolved, and visualized in a three-dimensional space. In sum, we found that there was little overlap in the distribution of VWF and fibrinogen in resting state platelets. In an important control, the distributions of green and red secondary antibodies overlapped completely when different color secondary antibodies directed against the same first antibody were used. Moreover, the same result was observed using different first antibodies and switching second antibody color to switch the color of VWF and fibrinogen staining. No accumulation of fibrinogen in late endosomes or lysosomes was detected by co-staining with LAMP2, a late endosome/lysosome membrane protein. Significantly, we found that in handling activated platelets there was differential retention of fibrinogen-positive granules relative to VWF positive granules. Our results indicate that VWF and fibrinogen are differentially packaged in human platelets. Moreover, the results suggest that differential packaging could support differential release of alpha-granule proteins.

Inhibition of Poly(ADP-Ribose) Polymerase Enhances Cell Death and Improves Tumor Growth Delay in Irradiated Lung Cancer Models

Poly(ADP-ribose) polymerase-1 (PARP-1) is the founding member of a family of enzymes that catalyze the addition of ADP-ribose units to proteins that mediate DNA repair pathways. Ionizing radiation induces DNA strand breaks, suggesting that PARP-1 inhibition may sensitize tumor cells to radiation. We investigated the combination of PARP-1 inhibition with radiation in lung cancer models. ABT-888, a novel potent PARP-1 inhibitor, was used to explore the effects of PARP-1 inhibition on irradiated tumors and tumor vasculature. ABT-888 reduced clonogenic survival in H460 lung cancer cells, and inhibited DNA repair as shown by enhanced expression of DNA strand break marker histone gamma-H2AX. Both apoptosis and autophagy contributed to the mechanism of increased cell death. Additionally, ABT-888 increased tumor growth delay at well-tolerated doses in murine models. For a 5-fold increase in tumor volume, tumor growth delay was 1 day for ABT-888 alone, 7 days for radiation alone, and 13.5 days for combination treatment. Immunohistochemical staining of tumor sections revealed an increase in terminal deoxyribonucleotide transferase-mediated nick-end labeling apoptotic staining, and a decrease in Ki-67 proliferative staining after combination treatment. Matrigel assay showed a decrease in in vitro endothelial tubule formation with ABT-888/radiation combination treatment, and von Willebrand factor staining of tumor sections revealed decreased vessel formation in vivo, suggesting that this strategy may also target tumor angiogenesis. We conclude that PARP-1 inhibition shows promise as an effective means of enhancing tumor sensitivity to radiation, and future clinical studies are needed to determine the potential of ABT-888 as a radiation enhancer.

PlexinD1 deficiency induces defects in axial skeletal morphogenesis

Axial patterning in embryonic skeletogenesis associates with coordinated programming of somitogenesis and angiogenesis. As seen in endochondral bone formation, skeletogenesis is closely related to angiogenesis during development. PlexinD1 is a member of plexin family, is expressed in central nervous system and endothelium, and plays a role in blood vessel patterning and endothelium positioning during embryonic development. Here, we examined the effects of PlexinD1 deficiency on skeletogenesis. Three-dimensional micro CT examination revealed that PlexinD1 deficiency resulted in axial skeletal patterning defects including malformation in vertebral body and rib bone shape. Histological examination of the vertebral bodies and long bones showed that PlexinD1 deficiency altered the development of cartilage. PlexinD1 deficiency did not affect the levels of von Willebrand factor staining in relatively large vessels not attached but close to the vertebral body of mice. However, PlexinD1 deficiency reduced the von Willebrand factor (vWf) staining in most of the microvasculatures attached to the vertebral bone. PlexinD1 was expressed in osteoblastic cells and bone tissues of newborn and adult mice. As most of the homozygous knockout mice did not survive, we examined the role of PlexinD1 in bone formation in heterozygous adult mice subjected to bone marrow ablation. However, PlexinD1 heterozygous knockout did not reveal defects in new bone formation. In conclusion, PlexinD1 is involved in the patterning of axial skeletogenesis.