Analysis of PDGF-AB and -BB in serum of intact and ovariectomized (OVX) pigs

Abstract only availablePrevious studies in our group demonstrated that terminal microvascular networks in dura mater of ovariectomized (OVX) pigs undergo significant remodeling characterized by a decrease in microvessel density, capillary rarefaction, and increase in blood vessel permeability. It was postulated that post OVX vascular remodeling is estrogen-dependent and could involve changes in expression levels of relevant growth factors and receptors on both systemic and local levels. Comparison of 41 relevant growth factors and receptors in serum of intact female and OVX animals using antibody array revealed most robust changes in the expression levels of platelet-derived growth factors (PDGF) -AB and -BB, both of which are potent regulators of growth and survival in a vascular tissue. To corroborate the data from the antibody array, we conducted SDS-Page and Western blot analysis using monoclonal antibody directed against B chain of PDGF, which recognizes both PDGF-AB and PDGF-BB. The Western blot analysis revealed several species of PDGF-AB and BB possibly existing in porcine serum, notably p24, p36 and p54, which are consistent with differing stages of posttranslational processing and maturation of PDGF. Densitometry analysis confirmed antibody array results showing significant decrease in PDGF-AB and PDGF-BB expression levels in post OVX animals compared to intact female swine. Our ongoing experiments aim at isolating and verifying specific bands, and analysis of the expression levels and autophosphorylation of PDGF receptors alpha and beta in different vascular compartments.Molecular Imaging Progra

Systemic and local changes in PDGF system associated with post ovariectomy microvascular remodeling in pigs [abstract]

Abstract only availablePrevious studies in our group demonstrated that terminal microvascular networks in dura mater of ovariectomized (OVX) pigs undergo significant remodeling characterized by a decrease in microvessel density, capillary rarefaction, and increase in blood vessel permeability. It was postulated that post OVX vascular remodeling is estrogen-dependent and could involve changes in the expression of relevant growth factors and receptors on both systemic and local levels. Systemically, comparison of growth factors and receptors in serum of intact female (IF) and OVX pigs using antibody array revealed most robust changes in expression levels of platelet-derived growth factors (PDGF) -AB and -BB, both of which are potent regulators of growth and survival in vascular tissue. These results were corroborated by Western blot analysis using monoclonal antibody directed against the B chain of PDGF, which recognizes both PDGF-AB and -BB. Densitometry analysis confirmed antibody array results showing a significant decrease in PDGF-AB and -BB expression levels in post OVX animals compared to IF swine. Lower levels of circulating PDGF could translate into a weakened response of systemic repair mechanisms during vascular damage in OVX animals. On a tissue level, however, two months post OVX there was a significant increase in local PDGF expression in OVX animals compared to IF swine accompanied by a corresponding increase in phosphorylation of PDGF receptor alpha. Our current hypothesis is that hypoxic stromal responses, triggered by initial microvessel loss in OVX animals, activate PDGF/VEGF system in an attempt to restore microvasculature via angiogenic processes. Ongoing studies are aimed at identifying other factors and pathways involved in the regulation of post OVX vascular remodeling.Life Sciences Undergraduate Research Opportunity Progra

Inhibition of Glycogen Synthase Kinase-3β Counteracts Ligand-Independent Activity of the Androgen Receptor in Castration Resistant Prostate Cancer

In order to generate genomic signals, the androgen receptor (AR) has to be transported into the nucleus upon androgenic stimuli. However, there is evidence from in vitro experiments that in castration-resistant prostate cancer (CRPC) cells the AR is able to translocate into the nucleus in a ligand-independent manner. The recent finding that inhibition of the glycogen-synthase-kinase 3β (GSK-3β) induces a rapid nuclear export of the AR in androgen-stimulated prostate cancer cells prompted us to analyze the effects of a GSK-3β inhibition in the castration-resistant LNCaP sublines C4-2 and LNCaP-SSR. Both cell lines exhibit high levels of nuclear AR in the absence of androgenic stimuli. Exposure of these cells to the maleimide SB216763, a potent GSK-3β inhibitor, resulted in a rapid nuclear export of the AR even under androgen-deprived conditions. Moreover, the ability of C4-2 and LNCaP-SSR cells to grow in the absence of androgens was diminished after pharmacological inhibition of GSK-3β in vitro. The ability of SB216763 to modulate AR signalling and function in CRPC in vivo was additionally demonstrated in a modified chick chorioallantoic membrane xenograft assay after systemic delivery of SB216763. Our data suggest that inhibition of GSK-3β helps target the AR for export from the nucleus thereby diminishing the effects of mislocated AR in CRPC cells. Therefore, inhibition of GSK-3β could be an interesting new strategy for the treatment of CRPC

Corneal Injury Is Associated With Stromal and Vascular Alterations Within Cranial Dura Mater

The cornea and cranial dura mater share sensory innervation. This link raises the possibility that pathological impulses mediated by corneal injury may be transmitted to the cranial dura, trigger dural perivascular/connective tissue nociceptor responses, and induce vascular and stromal alterations affecting dura mater blood and lymphatic vessel functionality. In this study, using a mouse model, we demonstrate for the first time that two weeks after the initial insult, alkaline injury to the cornea leads to remote pathological changes within the coronal suture area of the dura mater. Specifically, we detected significant pro-fibrotic changes in the dural stroma, as well as vascular remodeling characterized by alterations in vascular smooth muscle cell (VSMC) morphology, reduced blood vessel VSMC coverage, endothelial cell expression of the fibroblast specific protein 1, and significant increase in the number of podoplanin-positive lymphatic sprouts. Intriguingly, the deficiency of a major extracellular matrix component, small leucine-rich proteoglycan decorin, modifies both the direction and the extent of these changes. As the dura mater is the most important route for the brain metabolic clearance, these results are of clinical relevance and provide a much-needed link explaining the association between ophthalmic conditions and the development of neurodegenerative diseases

Continuous real time ex vivo epifluorescent video microscopy for the study of metastatic cancer cell interactions with microvascular endothelium

Recent studies suggest that only endothelium-attached malignant cells are capable of giving rise to hematogenous cancer metastases. Moreover, tumor cell adhesion to microvascular endothelium could be crucial in metastasis predilection to specific organs or tissues. However, the existing in vitro and in vivo techniques do not provide for sufficient delineation of distinct stages of a dynamic multi-step intravascular adhesion process. Here we report the development of an experimental system allowing for prolonged continuous ex vivo real-time observation of malignant cell adhesive interactions with perfused microvessels of a target organ in the context of its original tissue. Specifically, the vasculature of excised dura mater perfused with prostate cancer cells is described. An advantage of this technique is that selected fluorescently labeled tumor cells can be followed along identified vascular trees across the entire tissue specimen. The techniques provide for superior microvessel visualization and allow for uninterrupted monitoring and video recording of subsequent adhesion events such as rolling, docking (initial reversible adhesion), locking (irreversible adhesion), and flattening of metastatic cancer cells within perfused microvasculature on a single cell level. The results of our experiments demonstrate that intravascular adhesion of cancer cells differs dramatically from such of the leukocytes. Within dura microvessels perfused at physiological rate, non-interacting, floating, tumor cells move at velocities averaging 7.2×10 3  μm/s. Some tumor cells, similarly to leukocytes, exhibit rolling-like motion patterns prior to engaging into more stable adhesive interactions. In contrast, other neoplastic cells became stably adhered without rolling showing a rapid reduction in velocity from 2×10 3 to 0 μm/s within fractions of a second. The experimental system described herein, while developed originally for studying prostate cancer cell interactions with porcine dura mater microvasculature, offers great flexibility in adhesion experiments design and is easily adapted for use with a variety of other tissues including human.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42585/1/10585_2004_Article_5120583.pd

Estrogen-Dependent Changes in Dura Mater Microvasculature Add New Insights to the Pathogenesis of Headache

The pathogenesis of headaches is a matter of ongoing discussion of two major theories describing it either as a vascular phenomenon resulting from vasodilation or primarily as a neurogenic process accompanied by secondary vasodilation associated with sterile neurogenic inflammation. While summarizing current views on neurogenic and vascular origins of headache, this mini review adds new insights regarding how smooth muscle-free microvascular networks, discovered within dura mater connective tissue stroma (previously thought to be “avascular”), may become a site of initial insult generating the background for the development of headache. Deficiencies in estrogen-dependent control of microvascular integrity leading to plasma protein extravasation, potential activation of perivascular and connective tissue stroma nociceptive neurons, and triggering of inflammatory responses are described. Finally, possible avenues for controlling and preventing these pathophysiological changes are discussed

Mechanical Entrapment Is Insufficient and Intercellular Adhesion Is Essential for Metastatic Cell Arrest in Distant Organs

In this report, we challenge a common perception that tumor embolism is a size-limited event of mechanical arrest, occurring in the first capillary bed encountered by blood-borne metastatic cells. We tested the hypothesis that mechanical entrapment alone, in the absence of tumor cell adhesion to blood vessel walls, is not sufficient for metastatic cell arrest in target organ microvasculature. The in vivo metastatic deposit formation assay was used to assess the number and location of fluorescently labeled tumor cells lodged in selected organs and tissues following intravenous inoculation. We report that a significant fraction of breast and prostate cancer cells escapes arrest in a lung capillary bed and lodges successfully in other organs and tissues. Monoclonal antibodies and carbohydrate-based compounds (anti-Thomsen-Friedenreich antigen antibody, anti-galectin-3 antibody, modified citrus pectin, and lactulosyl-l-leucine), targeting specifically β-galactoside-mediated tumor-endothelial cell adhesive interactions, inhibited by >90% the in vivo formation of breast and prostate carcinoma metastatic deposits in mouse lung and bones. Our results indicate that metastatic cell arrest in target organ microvessels is not a consequence of mechanical trapping, but is supported predominantly by intercellular adhesive interactions mediated by cancer-associated Thomsen-Friedenreich glycoantigen and β-galactoside-binding lectin galectin-3. Efficient blocking of β-galactoside-mediated adhesion precludes malignant cell lodging in target organs

Inhibition of Prostate Cancer Bone Metastasis by Synthetic TF Antigen Mimic/Galectin-3 Inhibitor Lactulose-l-Leucine1

Currently incurable, prostate cancer metastasis has a remarkable ability to spread to the skeleton. Previous studies demonstrated that interactions mediated by the cancer-associated Thomsen-Friedenreich glycoantigen (TF-Ag) and the carbohydrate-binding protein galectin-3 play an important role in several rate-limiting steps of cancer metastasis such as metastatic cell adhesion to bone marrow endothelium, homotypic tumor cell aggregation, and clonogenic survival and growth. This study investigated the ability of a synthetic small-molecular-weight nontoxic carbohydrate-based TF-Ag mimic lactulose-l-leucine (Lac-l-Leu) to inhibit these processes in vitro and, ultimately, prostate cancer bone metastasis in vivo. Using an in vivo mouse model, based on intracardiac injection of human PC-3 prostate carcinoma cells stably expressing luciferase, we investigated the ability of Lac-l-Leu to impede the establishment and growth of bone metastasis. Parallel-flow chamber assay, homotypic aggregation assay, modified Boyden chamber assay, and clonogenic growth assay were used to assess the effects of Lac-l-Leu on tumor cell adhesion to the endothelium, homotypic tumor cell aggregation, transendothelial migration, and clonogenic survival and growth, respectively. We report that daily intraperitoneal administration of Lac-l-Leu resulted in a three-fold (P < .05) decrease in metastatic tumor burden compared with the untreated control. Mechanistically, the effect of Lac-l-Leu, which binds and inhibits galectins by mimicking essential structural features of the TF-Ag, was associated with a dose-dependent inhibition of prostate cancer cell adhesion to bone marrow endothelium, homotypic aggregation, transendothelial migration, and clonogenic growth. We conclude that small-molecular-weight carbohydrate-based compounds targeting β-galactoside-mediated interactions could provide valuable means for controlling and preventing metastatic prostate cancer spread to the skeleton