Collagen I triggers directional migration, invasion and matrix remodeling of stroma cells in a 3D spheroid model of endometriosis.

Endometriosis is a painful gynecological condition characterized by ectopic growth of endometrial cells. Little is known about its pathogenesis, which is partially due to a lack of suitable experimental models. Here, we use endometrial stromal (St-T1b), primary endometriotic stromal, epithelial endometriotic (12Z) and co-culture (1:1 St-T1b:12Z) spheroids to mimic the architecture of endometrium, and either collagen I or Matrigel to model ectopic locations. Stromal spheroids, but not single cells, assumed coordinated directional migration followed by matrix remodeling of collagen I on day 5 or 7, resembling ectopic lesions. While generally a higher area fold increase of spheroids occurred on collagen I compared to Matrigel, directional migration was not observed in co-culture or in 12Z cells. The fold increase in area on collagen I was significantly reduced by MMP inhibition in stromal but not 12Z cells. Inhibiting ROCK signalling responsible for actomyosin contraction increased the fold increase of area and metabolic activity compared to untreated controls on Matrigel. The number of protrusions emanating from 12Z spheroids on Matrigel was decreased by microRNA miR-200b and increased by miR-145. This study demonstrates that spheroid assay is a promising pre-clinical tool that can be used to evaluate small molecule drugs and microRNA-based therapeutics for endometriosis.

Laminin degradation by matrix metalloproteinase 9 promotes ketamine-induced neuronal apoptosis in the early developing rat retina.

AIMS: During early development, laminin degradation contributes to the death of neurons. This study aims to investigate the role and regulation of laminin in ketamine-induced apoptosis. METHODS: We performed terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) and immunohistochemical assays to investigate the roles of the non-integrin laminin receptor, matrix metalloproteinase 9 (MMP9) in ketamine-induced neuronal apoptosis. In situ zymography, Western blot, and immunofluorescence were used to explore the relationships between laminin, MMP9 activity, and Zn2+ . Experiments were performed using whole-mount retinas dissected from Sprague Dawley rats. RESULTS: The TUNEL and immunohistochemical assays indicated that ketamine-induced neuronal apoptosis in early developing rat retina. Blockade of non-integrin laminin receptor promoted ketamine-induced apoptosis, while non-integrin laminin receptor activation attenuated ketamine-induced apoptosis. Ketamine-induced laminin degradation, possibly by enhancing the activity of MMP9. MMP9 inhibition reduced ketamine-induced apoptosis by reducing laminin degradation. Downregulation of Zn2+ attenuated the increased MMP9 activity, laminin degradation caused by ketamine and significantly reduced ketamine-induced neuronal apoptosis. CONCLUSION: Laminin degradation by MMP9 promoted ketamine-induced neuronal apoptosis in early developing rat retina. The non-integrin laminin receptor may be a pathway involved in ketamine-induced apoptosis. Zn2+ downregulation may play a protective role against ketamine-induced neuronal apoptosis through inhibiting MMP9 activity.

Cultivated Orostachys japonicus extract inhibits VEGF-induced angiogenesis via regulation of VEGFR2 signaling pathway in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Orostachys japonicus A. Berger (O. japonicus), so-called Wa-song in Korea, a traditional food and medicine that grows on mountain rocks and roof tiles. Wa-song containing various phenolic compounds have been reported as a medicinal plant for prevention of fibrosis, cancer, inflammation, and oxidative damage. AIM OF THE STUDY: The present study was designed to examine the anti-angiogenic effects of cultivated Orostachys japonicus 70% ethanol extract (CE) in vascular endothelial growth factor (VEGF)-stimulated human umbilical vein endothelial cells (HUVECs). MATERIALS AND METHODS: CE was prepared with 70% ethanol. HUVECs, rat aortic rings, and matrigel plug in mice were treated with CE (10-20 µg/mL) and VEGF (20-50 ng/mL), and the anti-angiogenic activities of CE were analyzed by SRB, wound healing, trans-well invasion, capillary-like tubule formation, rat aortas, Western blot, and matrigel plug assay. Phenolic compounds in CE were analyzed using a high-performance liquid chromatography (HPLC)-PDA system. RESULTS: Treatment of CE (10-20 µg/mL) markedly suppressed proliferation of HUVECs in the presence (from 136.5% to 112.2%) or absence of VEGF (from 100.0% to 92.1%). The proliferation inhibitory effect of CE was caused by G0/G1 cell cycle arrest, and the decrease of CDK-2, CDK-4, Cyclin D1 and Cyclin E1. Furthermore, CE treatment showed significant angiogenesis inhibitory effects on motility, invasion and micro-vessel formation of HUVECs, rat aortic rings and subcutaneous matrigels under VEGF-stimulation condition. In HUVECs, CE-induced anti-angiogenic effect was regulated by inhibition of the PI3K/AKT/mTOR, MAPK/p38, MAPK/ERK, FAK-Src, and VEGF-VEGFR2 signaling pathways. CONCLUSION: This study demonstrated that CE might be used as a potential natural substance, multi-targeted angiogenesis inhibitor, functional food material.

Angioedema and Hemorrhage After 4.5-Hour tPA (Tissue-Type Plasminogen Activator) Thrombolysis Ameliorated by T541 via Restoring Brain Microvascular Integrity.

Background and Purpose- tPA (tissue-type plasminogen activator) is the only recommended intravenous thrombolytic agent for ischemic stroke. However, its application is limited because of increased risk of hemorrhagic transformation beyond the time window. T541 is a Chinese compound medicine with potential to attenuate ischemia and reperfusion injury. This study was to explore whether T541-benefited subjects underwent tPA thrombolysis extending the time window. Methods- Male C57BL/6 N mice were subjected to carotid artery thrombosis by stimulation with 10% FeCl3 followed by 10 mg/kg tPA with/without 20 mg/kg T541 intervention at 4.5 hours. Thrombolysis and cerebral blood flow were observed dynamically until 24 hours after drug treatment. Neurological deficit scores, brain edema and hemorrhage, cerebral microvascular junctions and basement membrane proteins, and energy metabolism in cortex were assessed then. An in vitro hypoxia/reoxygenation model using human cerebral microvascular endothelial cells was used to evaluate effect of T541 on tight junctions and F-actin in the presence of tPA. Results- tPA administered at 4.5 hours after carotid thrombosis resulted in a decrease in thrombus area and survival rate, whereas no benefit on cerebral blood flow. Study at 24 hours after tPA administration revealed a significant angioedema and hemorrhage in the ischemia hemisphere, a decreased expression of junction proteins claudin-5, zonula occludens-1, occludin, junctional adhesion molecule-1 and vascular endothelial cadherin, and collagen IV and laminin. Meanwhile, ADP/ATP, AMP/ATP, and ATP5D (ATP synthase subunit) expression and activities of mitochondria complex I, II, and IV declined, whereas malondialdehyde and 8-Oxo-2'-deoxyguanosine increased and F-actin arrangement disordered. All the insults after tPA treatment were attenuated by addition of T541 dose dependently. Conclusions- The results suggest T541 as a potential remedy to attenuate delayed tPA-related angioedema and hemorrhage and extend time window for tPA treatment. The potential of T541 to upregulate energy metabolism and protect blood-brain barrier is likely attributable to its effects observed.

Blistering and Skin Fragility Due to Imatinib Therapy: Loss of Laminin and Collagen IV as a Possible Cause of Cutaneous Basement Membrane Instability.

Imatinib mesylate (Glivec; Novartis AG, Basel, Switzerland) is a tyrosine kinase inhibitor which is used in the treatment of oncologic diseases like chronic myeloid leukemia and gastrointestinal stroma tumor (GIST). Among cutaneous side effects, bullous reactions are rare. The authors describe the case of a 66-year-old woman developing blistering and skin fragility on her hands, foot, lower legs, and back after intake of imatinib for treatment of GIST. Biopsy showed vacuolar alteration at the dermoepidermal junction (DEJ) associated with a few lymphocytes and a subepidermal blister. The upper papillary dermis below the vacuolar alteration and below the blister showed hyalinization and loss of elastic microfibrils. Direct immunofluorescence was negative for deposits of immunoglobulins. Immunofluorescence on cryosections revealed loss of laminin and collagen IV in vacuoles at the DEJ. Electron microscopy showed dissolution of lamina lucida and lamina densa of the basement membrane below as well as next to the vacuoles and blister. In conclusion, the authors present the first patient with GIST with blistering and skin fragility due to imatinib therapy. As a pathophysiological explanation the authors propose loss of laminin and collagen IV at the DEJ leading to basement membrane instability and blistering. This case also suggests additional features reminiscent of lichen sclerosus induced by imatinib, a drug which is actually known for its antifibrotic effects.

Growth hormone in the presence of laminin modulates interaction of human thymic epithelial cells and thymocytes in vitro.

BACKGROUND: Several evidences indicate that hormones and neuropeptides function as immunomodulators. Among these, growth hormone (GH) is known to act on the thymic microenvironment, supporting its role in thymocyte differentiation. The aim of this study was to evaluate the effect of GH on human thymocytes and thymic epithelial cells (TEC) in the presence of laminin. RESULTS: GH increased thymocyte adhesion on BSA-coated and further on laminin-coated surfaces. The number of migrating cells in laminin-coated membrane was higher in GH-treated thymocyte group. In both results, VLA-6 expression on thymocytes was constant. Also, treatment with GH enhanced laminin production by TEC after 24 h in culture. However, VLA-6 integrin expression on TEC remained unchanged. Finally, TEC/thymocyte co-culture model demonstrated that GH elevated absolute number of double-negative (CD4(-)CD8(-)) and single-positive CD4(+) and CD8(+) thymocytes. A decrease in cell number was noted in double-positive (CD4(+)CD8(+)) thymocytes. CONCLUSIONS: The results of this study demonstrate that GH is capable of enhancing the migratory capacity of human thymocytes in the presence of laminin and promotes modulation of thymocyte subsets after co-culture with TEC.

Growth hormone in the presence of laminin modulates interaction of human thymic epithelial cells and thymocytes in vitro

BACKGROUND: Several evidences indicate that hormones and neuropeptides function as immunomodulators. Among these, growth hormone (GH) is known to act on the thymic microenvironment, supporting its role in thymocyte differentiation. The aim of this study was to evaluate the effect of GH on human thymocytes and thymic epithelial cells (TEC) in the presence of laminin. RESULTS: GH increased thymocyte adhesion on BSA-coated and further on laminin-coated surfaces. The number of migrating cells in laminin-coated membrane was higher in GH-treated thymocyte group. In both results, VLA-6 expression on thymocytes was constant. Also, treatment with GH enhanced laminin production by TEC after 24 h in culture. However, VLA-6 integrin expression on TEC remained unchanged. Finally, TEC/thymocyte co-culture model demonstrated that GH elevated absolute number of double-negative (CD4-CD8-) and single-positive CD4+ and CD8+ thymocytes. A decrease in cell number was noted in double-positive (CD4+CD8+) thymocytes. CONCLUSIONS: The results of this study demonstrate that GH is capable of enhancing the migratory capacity of human thymocytes in the presence of laminin and promotes modulation of thymocyte subsets after co-culture with TEC.

Effects of penicillin and erythromycin on adherence of invasive and noninvasive isolates of Streptococcus pyogenes to laminin.

This study investigated the possible relationship between the invasiveness of group A Streptococcus (GAS) strains and their abilities to adhere to laminin and assessed the effects of subinhibitory concentrations of penicillin and erythromycin on the ability of GAS to adhere to laminin. The adherence of noninvasive and highly invasive isolates of GAS to laminin was significantly higher than the adherence displayed by isolates of low invasiveness. Antibiotic treatment caused significant reductions in adherence to laminin in all three groups of strains. Penicillin was more successful in reducing the adherence abilities of the tested GAS strains than erythromycin.

Effects of penicillin and erythromycin on adherence of invasive and noninvasive isolates of Streptococcus pyogenes to laminin

This study investigated the possible relationship between the invasiveness of group A Streptococcus (GAS) strains and their abilities to adhere to laminin and assessed the effects of subinhibitory concentrations of penicillin and erythromycin on the ability of GAS to adhere to laminin. The adherence of noninvasive and highly invasive isolates of GAS to laminin was significantly higher than the adherence displayed by isolates of low invasiveness. Antibiotic treatment caused significant reductions in adherence to laminin in all three groups of strains. Penicillin was more successful in reducing the adherence abilities of the tested GAS strains than erythromycin.

[Clinical evaluation of ocriplasmin as a vitreolytic agent].

Incomplete perifoveal posterior vitreous detachment (PVD) associated with abnormal vitreomacular adhesion (VMA) can cause vitreomacular traction (VMT) and macular hole (MH) formation, which require vitrectomy treatment. Pharmacologic vitreolysis, which is intravitreal injection with vitreolytic enzymes to resolve VMA, may be used as an alternative therapy.Ocriplasmin, formerly known as microplasmin, is a recombinant truncated form of plasmin with proteolytic activity against fibronectin and laminin.It was recently approved for VMA treatment in the European Union and USA. Phase III studies indicated that ocriplasmin injection was a safe and effective treatment for selected cases of symptomatic VMA and MH. VMA release was achieved in 26.5% of ocriplasmin-injected eyes versus 10.1% of the placebo group. MH closure was achieved in 40.6% as compared with 10.6% of the placebo group.In comparison with the outcome after vitrectomy, the success rate of ocriplasmin was still far below expectation. Ocular adverse events included vitreous floaters, photopsia and profound visual decline. Its efficacy and safety need to be further evaluated in more clinical trials.

TCDD disrupts posterior palatogenesis and causes cleft palate.

Dioxins (e.g. 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) cause cleft palate at a high rate. A post-fusional split may contribute to the pathogenesis, and tissue fragility may be a concern. The objective of this study was to investigate the effects of TCDD on the palatal epithelium, bone and muscle, which contribute to tissue integrity. ICR mice (10-12 weeks old) were used. TCDD was administered on E12.5 at 40 mg/kg. Immunohistochemical staining for AhR, ER-α, laminin, collagen IV, osteopontin, Runx2, MyoD, and desmin were performed. Furthermore, western blot analysis for osteopontin, Runx2, MyoD, and desmin were performed to evaluate protein expression in the palatal tissue. Immunohistologically, there was little difference in the collagen IV and laminin localization in the palatal epithelium between control versus TCDD-treated mice. Runx2 and osteopontin immunoreactivity decreased in the TCDD-treated palatal bone, and MyoD and desmin decreased in the TCDD-treated palatal muscle. AhR and ER-α immunoreactivity were localized to the normal palatal bone, but ER-α was diminished in the TCDD-treated palate. On western blot analysis, Runx2, MyoD, and desmin were all downregulated in the TCDD-treated palate. TCDD may suppress palatal osteogenesis and myogenesis via AhR, and cause cleft palates via a post-fusional split mechanism, in addition to a failure of palatal fusion.

Amphiphilic suramin dissolves Matrigel, causing an 'inhibition' artefact within in vitro angiogenesis assays.

The field of study concerning promotion and/or inhibition of angiogenesis has gathered much attention in the scientific community. A great deal of work has been invested towards defining reproducible assays to gauge for promotion or inhibition of angiogenesis in response to drug treatments or growth conditions. Two common components of these assays were noted by our group to have an unexpected and previously unreported interaction. Suramin is a commercially available compound, commonly used as a positive control for in vitro angiogenic inhibition assays. Matrigel is a popular extracellular substrate that supports angiogenic network formation when endothelial cells are cultured on its surface. However, our group demonstrated that suramin alone (without the presence of cells) will actively dissolve Matrigel, causing the extracellular matrix to transition from the gel-like physical state to a more liquid state. This causes cells on the Matrigel to congregate and sink to the bottom of the well. Therefore, previous observations of inhibition of endothelial cell angiogenesis through the incubation with suramin (including previous observations made by our group) are, largely, an artefact caused by suramin and matrix interaction rather than suramin and cells interaction, as previously reported. Our results suggest that the presence of sulphate groups and amphiphilic properties of suramin are likely responsible for the disruption of the matrix layer. We believe that this information is of prime importance to anyone using similar in vitro models, or employing suramin in any therapy or drug development assays.

Anti-angiogenic properties of carnosol and carnosic acid, two major dietary compounds from rosemary.

BACKGROUND: The use of rosemary (Rosmarinus officinalis) leaves and their constituents as a source of dietary antioxidants and flavoring agents is continuously growing. Carnosol and carnosic acid, two major components of rosemary extracts, have shown activity for cancer prevention and therapy. AIM OF THE STUDY: In this study, we investigate the cytotoxic and anti-angiogenic activities of carnosol and carnosic acid, in order to get further insight into their mechanism of action. RESULTS: Our results demonstrate that the mentioned diterpenes inhibit certain functions of endothelial cells, namely, differentiation, proliferation, migration and proteolytic capability. Our data indicate that their growth inhibitory effect, exerted on proliferative endothelial and tumor cells, could be due to, at least in part, an induction of apoptosis. Inhibition of the mentioned essential steps of in vitro angiogenesis agrees with the observed inhibition of the in vivo angiogenesis, substantiated by using the chick chorioallantoic membrane assay. CONCLUSIONS: The anti-angiogenic activity of carnosol and carnosic acid could contribute to the chemopreventive, antitumoral and antimetastatic activities of rosemary extracts and suggests their potential in the treatment of other angiogenesis-related malignancies.

Exosomes released by K562 chronic myeloid leukemia cells promote angiogenesis in a Src-dependent fashion.

Exosomes, microvesicles of endocytic origin released by normal and tumor cells, play an important role in cell-to-cell communication. Angiogenesis has been shown to regulate progression of chronic myeloid leukemia (CML). The mechanism through which this happens has not been elucidated. We isolated and characterized exosomes from K562 CML cells and evaluated their effects on human umbilical endothelial cells (HUVECs). Fluorescent-labeled exosomes were internalized by HUVECs during tubular differentiation on Matrigel. Exosome localization was perinuclear early in differentiation, moving peripherally in cells undergoing elongation and connection. Exosomes move within and between nanotubular structures connecting the remodeling endothelial cells. They stimulated angiotube formation over a serum/growth factor-limited medium control, doubling total cumulative tube length (P = 0.003). Treatment of K562 cells with two clinically active tyrosine kinase inhibitors, imatinib and dasatinib, reduced their total exosome release (P < 0.009); equivalent concentrations of drug-treated exosomes induced a similar extent of tubular differentiation. However, dasatinib treatment of HUVECs markedly inhibited HUVEC response to drug control CML exosomes (P < 0.002). In an in vivo mouse Matrigel plug model angiogenesis was induced by K562 exosomes and abrogated by oral dasatinib treatment (P < 0.01). K562 exosomes induced dasatinib-sensitive Src phosphorylation and activation of downstream Src pathway proteins in HUVECs. Imatinib was minimally active against exosome stimulation of HUVEC cell differentiation and signaling. Thus, CML cell-derived exosomes induce angiogenic activity in HUVEC cells. The inhibitory effect of dasatinib on exosome production and vascular differentiation and signaling reveals a key role for Src in both the leukemia and its microenvironment.

In vivo and in vitro antitumor effects of nutrient mixture in murine leukemia cell line P-388.

AIM: Leukemia is characterized by uncontrolled marrow cell proliferation and metastatic foci. We investigated the antitumor potential of a nutrient mixture on malignant leukemia P-388 cells. METHODS: The nutrient mixture containing lysine, proline, ascorbic acid, green tea extract and other nutrients is formulated to target key pathways in cancer progression. The cells were treated with the mixture, and tested at doses 0, 10, 50, 100, 500 and 1000 µg/ml in triplicates. The effects were evaluated by cell proliferation, Matrigel invasion, cell morphology and apoptosis. The in vivo effect was measured in male nude mice (n = 12) inoculated with P-388 cells. After randomly dividing in two groups, each group was fed regular and the nutrient mixture supplemented diet and the mice were sacrificed after four weeks. RESULTS: The nutrient mixture decreased P-388 cell proliferation at 500 and 1000 µg/ml. Only 10% cells were viable at 1000 µg/ml. Matrigel invasion was significantly inhibited in a dose dependent manner with virtually total inhibition at 1000 µg/ml. Cell morphological features notably changed with dose increase to 1000 µg/ml. Analysis of apoptotic cells on live green caspase kit exhibited gradual increase with the increasing dose of the nutrient mixture, and at 1000 µg/ ml 92% of P-388 cells were in late apoptosis. Tumors in the group of mice supplemented with the nutrient mixture had 50% lower weight compared to the tumors in control group (p = 0.0105). Histopathologically, both the groups of tumors were similar, yet size of tumors in the group treated with the nutrient mixture was considerably smaller. CONCLUSION: These results indicate that the nutrient mixture exhibited significant action against multiple targets in P-388 leukemia and may have potential in human leukemia.

In vitro suppression of quercetin on hypertrophy and extracellular matrix accumulation in rat glomerular mesangial cells cultured by high glucose.

Quercetin's protective effects on the glomerulosclerosis of diabetic nephropathy (DN) in rat mesangial cells were investigated. The cell cycles, type IV collagen and laminin, TGF-ß(1) mRNA, Smad 2/3 and Smad 7, and activities of cell antioxidases were measured. Compared with the high glucose group, quercetin may decrease the cell percentages of G(0)/G(1) phase, Smad 2/3 expression, laminin and type IV collagen, and TGF-ß(1) mRNA level significantly. The antioxidant capacity, the cell percentages of S phase and Smad 7 expression was significantly increased by quercetin. These results suggest that quercetin is a protective agent against glomerulosclerosis in DN.

Kainic acid and 3-Nitropropionic acid induced expression of laminin in vascular elements of the rat brain.

Laminin is a glycoprotein component of the basement membrane and has been reported to be found in different areas of the nervous system including brain endothelial cells, Schwann cells and peripheral nerves. Although the in-vitro studies suggest that laminin plays an important role in growth and neurite extension of cultured neurons, localization of laminin in the brain has been controversial and inconsistent results have been reported. Recently, laminin immunoreactivity has been used as a marker for vascular elements in the brain. In this study, we have investigated the effect of two mechanistically different neurotoxins, kainic acid (KA), an NMDA agonist and 3-Nitropropionic acid (3-NPA), an inhibitor of mitochondrial respiration, on brain vascular elements revealed by laminin immunolabeling. We also explored whether administration of these two neurotoxic drugs correlate with the neuronal degeneration observed after neurotoxic insult by staining with Fluoro-Jade C dye. We have employed single immunolabeling to localize laminin in the brains. In KA treated rats, most of the laminin immunoreactivity is present in the piriform cortex, corpus callosum (myelinated tracts) amygdala, hippocampus, ventral thalamus and tenia tacta. In 3-NPA treated animals, laminin immunoreactivity was confined mostly to the striatum. In contrast, saline treated rats showed very little laminin immunolabeling around capillaries, arteries and in the meningeal membranes. To determine the effects of these neurotoxins on the integrity of the blood brain barrier (BBB), endothelial brain barrier antigen (EBA) immunolabeling was also performed. In addition, we performed CD11b immunolabeling to evaluate the effect of 3-NPA and KA on the activation of microglia in the brain. CD11b was dramatically increased in KA and 3-NPA treated animals. We have also combined laminin immunolabeling with Fluoro-Jade C labeling to evaluate the spatio-temporal association of degenerating neurons and the expression of laminin containing microvessels. Areas which showed intense laminin immunolabeling following KA or 3-NPA exposure correlated with those exhibiting the greatest number of degenerating neurons observed after Fluoro-Jade C staining. EBA-laminin double immunolabeling demonstrated that the expressions of laminin were predominantly localized in the areas (cortex, thalamus and hippocampus) where EBA has been either reduced or is absent. Our results from these experiments demonstrate that vascular laminin expression increases after treatment with KA or 3-NPA, suggesting the occurrence of neovascularization. Microglia may also contribute to the neurotoxic induced neovascularization and neurodegeneration.

Differential effects of sulfated triterpene glycosides, holothurin A1, and 24-dehydroechinoside A, on antimetastasic activity via regulation of the MMP-9 signal pathway.

Two sulfated triterpene glycosides, holothurin A(1) (HA(1)) and 24-dehydroechinoside A (DHEA), isolated from the sea cucumber Pearsonothuria graeffei, are of the holostane type with 18(20)-lactone and identical carbohydrate chains. DHEA has a side chain 23 (24)-double bond, while HA(1) has a hydroxyl group at C-21. In this study, we compared the effects of DHEA and HA(1) on metastasis in vitro and in vivo. The results show that HA(1) and DHEA treatment significantly suppressed adhesion of human hepatocellular liver carcinoma cells (HepG2) to both matrigel and human endothelial cells (ECV-304) and inhibited HepG2 cell migration and invasion in a dose-dependant manner. HA(1) and DHEA reduced tube formation of ECV-304 cells on the matrigel in vitro and attenuated neovascularization in the chick embryo using the chorioallantoic membrane (CAM) assay in vivo. Immunocytochemistry analyses revealed that both HA(1) and DHEA significantly decreased the expression of the matrix metallo-proteinase-9 (MMP-9) and increased the expression level of tissue inhibitor of metalloproteinase-1 (TIMP-1), an important regulator of MMP-9 activation. Western blot analyses demonstrated that HA(1) and DHEA remarkably abolished the expression of vascular endothelial growth factor (VEGF). The expression of nuclear factor-kappa B (NF-κB) was significantly decreased by HA(1), while DHEA treatment had no effect on the down regulation of NF-κB expression. These data suggest that both DHEA and HA(1) exert significant antimetastatic activities by inhibiting MMP-9 and VEGF expression. DHEA-induced antimetastasis was more potent than HA(1). In addition, only HA(1) treatment downregulated the expression level of NF-κB, suggesting that the antimetastatic activity of triterpene glycosides derived from P. graeffei can be either NF-κB-dependent or -independent, depending on their structure.

Microplasmin degrades fibronectin and laminin at vitreoretinal interface and outer retina during enzymatic vitrectomy.

PURPOSE: To determine whether intravitreal administration of microplasmin (microPlm) will degrade fibronectin (FN) and laminin (LN) in rat retina during microPlm-induced posterior vitreous detachment (PVD). METHODS: Increasing doses of microPlm, from 0.01 U to 0.03 U, were injected into the left eyes of 60 Sprague-Dawley rats to induce PVD. The right eyes were injected with the same volume of balanced salt solution (BSS). Histochemistry, scanning electron microscopy (SEM), and phase contrast microscopy were performed after 1 day and 7 days, to assess the remnant vitreous cortex. The FN and LN level located at the vitreoretinal interface and the outer retina were detected by immunohistochemistry. RESULTS: microPlm induced complete PVD in a dose-dependent fashion, without internal limiting membrane (ILM) damage (P = 0.0001, r = -0.479). The FN and LN in the photoreceptor cell layer (PCL) were completely degraded in all microPlm-treated eyes. In eyes with complete PVD, the FN, but not the LN, was completely removed from the ILM by microPlm treatment. CONCLUSION: Intravitreal injection of microplasmin degraded FN and LN at the vitreoretinal junction as well as at the outer retina.

Role of Ceacam1 in VEGF induced vasculogenesis of murine embryonic stem cell-derived embryoid bodies in 3D culture.

CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1), a type I transmembrane glycoprotein involved in cell-cell adhesion has been shown to act as an angiogenic factor for mouse and human endothelial cells. Based on the ability of CEACAM1 to initiate lumen formation in human mammary epithelial cells grown in 3D culture (Matrigel), we hypothesized that murine CEACAM1 may play a similar role in vasculogenesis. In order to test this hypothesis, murine embryonic stem (ES) cells stimulated with VEGF were differentiated into embryoid bodies (EB) for 8 days (-8-0 d) and transferred to Matrigel in the presence or absence of anti-CEACAM1 antibody for an additional 12 days (0-12 d). In the absence of anti-CEACAM1 antibody or in the presence of an isotype control antibody, the EB in Matrigel underwent extensive sprouting, generating lengthy vascular structures with well-defined lumina as demonstrated by confocal microscopy, electron microscopy, and immunohistochemical analysis. Both the length and architecture of the vascular tubes were inhibited by anti-CEACAM1 mAb CC1, a mAb that blocks the cell-cell adhesion functions of CEACAM1, thus demonstrating a critical role for this cell-cell adhesion molecule in generating and maintaining vasculogenesis. QRT-PCR analysis of the VEGF treated ES cells grown under conditions that convert them to EB revealed expression of Ceacam1 as early as -5 to -3 d reaching a maximum at day 0 at which time EBs were transferred to Matrigel, thereafter levels at first declined and then increased over time. Other markers of vasculogenesis including Pecam1, VE-Cad, and Tie-1 were not detected until day 0 when EBs were transferred to Matrigel followed by a steady increase in levels, indicating later roles in vasculogenesis. In contrast, Tie-2 and Flk-1 (VEGFR2) were detected on day five of EB formation reaching a maximum at day 0 on transfer to Matrigel, similar to Ceacam1, but after which Tie-2 declined over time, while Flk-1 increased over time. QRT-PCR analysis of the anti-CEACAM1 treated ES cells revealed a significant decrease in the expression of Ceacam1, Pecam1, Tie-1, and Flk-1, while VE-Cad and Tie-2 expression were unaffected. These results suggest that the expression and signaling of CEACAM1 may affect the expression of other factors known to play critical roles in vasculogenesis. Furthermore this 3D model of vasculogenesis in an environment of extracellular matrix may be a useful model for comparison to existing models of angiogenesis.