Piotr Religa

Influence of intraluminal thrombus on structural and cellular composition of abdominal aortic aneurysm wall

INTRODUCTION It has been suggested that the intraluminal thrombus of abdominal aortic aneurysm (AAA) affects the underlying vessel wall. Aneurysm enlargement has been associated with growth of thrombus, and rupture has been proposed to occur after bleeding into the thrombus. To examine how thrombus affects the vessel wall, we compared the morphology of aneurysm wall covered with thrombus with wall segments exposed to flowing blood. Material and methods Sixteen patients (14 men, 2 women; age range, 56-79 years) undergoing elective repair of AAA, where computed tomography scans showed thrombus and segments of the aneurysm wall exposed to flowing blood, were included in the study. Specimens from the aneurysm were taken for light and electron microscopy. Masson trichrome staining was performed for wall thickness determination and demonstration of collagen, and Weigert-van Gieson staining for elastin. The cellular composition was analyzed by immunohistochemistry with antibodies against CD3 for T cells, CD4 for T helper cells, CD8 for T cytotoxic cells, CD20 for B cells, CD68 for macrophages, and smooth muscle alpha-actin for smooth muscle cells (SMCs). Caspase-3 staining and TUNEL analysis were performed to evaluate apoptosis. RESULTS The aneurysm wall covered with thrombus was thinner and contained fewer elastin fibers, and the few that were found were often fragmented. This part of the wall also contained fewer SMCs and more apoptotic nuclei than the wall exposed to flowing blood. Clusters of inflammatory cells were detected in the media of the aneurysm wall and in higher numbers in the parts covered with thrombus. Electron microscopy showed that the aneurysm wall without thrombus contained a dense collagenous matrix with differentiated SMCs. In the segment covered with thrombus, SMCs were more dedifferentiated (synthetic) and apoptotic or necrotic. There were also an increased number of inflammatory cells located in close contact with SMCs in various stages of apoptosis. CONCLUSION The aneurysm wall covered with thrombus is thinner and shows more frequent signs of inflammation, apoptosis of SMCs, and degraded extracellular matrix. These findings suggest that thrombus formation and accumulation of inflammatory cells may perturb the structural integrity and stability of the vessel wall and thereby increase the risk for aneurysm rupture.

Vascular endothelial growth factor-a promotes peritumoral lymphangiogenesis and lymphatic metastasis.

Metastases are commonly found in the lymphatic system. The molecular mechanism of lymphatic metastasis is, however, poorly understood. Here we report that vascular endothelial growth factor (VEGF)-A stimulated lymphangiogenesis in vivo and that overexpression of VEGF-A in murine T241 fibrosarcomas induced the growth of peritumoral lymphatic vessels, which occasionally penetrated into the tumor tissue. As a result of peritumoral lymphangiogenesis, metastases in lymph nodes of mice were detected. VEGF-A-overexpressing tumors contained high numbers of infiltrating inflammatory cells such as macrophages, which are known to express VEGF receptor (VEGFR)-1. It seemed that in the mouse cornea, VEGF-A stimulated lymphangiogenesis through a VEGF-C/-D/VEGFR-3-independent pathway as a VEGFR-3 antagonist selectively inhibited VEGF-C-induced, but not VEGF-A-induced, lymphangiogenesis. Our data show that VEGF-A contributes to lymphatic mestastasis. Thus, blockage of VEGF-A-induced lymphangiogenesis may provide a novel approach for prevention and treatment of lymphatic metastasis.

Smooth-muscle progenitor cells of bone marrow origin contribute to the development of neointimal thickenings in rat aortic allografts and injured rat carotid arteries.

This study indicates that circulating progenitors of bone marrow origin give rise to cells with smooth muscle-like properties during formation of neointimal thickenings in the arterial wall after allotransplantation and after balloon injury. A segment of abdominal aorta was transplanted from female F344 to male LEW rats, and the grafts were analyzed for male cells by using the SRY gene as a marker. Immunostaining demonstrated that CD45-positive leukocytes made up 35–45% of the neointimal cells during the 8-week period examined. Concurrently, up to 70% of the neointimal cells were of host origin, as shown by real-time polymerase chain reaction for the SRY gene (Y chromosome). This suggests that the neointima contained host cells also of noninflammatory character. Accordingly, many cells positive for smooth-muscle &agr;-actin were detected in this layer. To explore the possible bone marrow origin of allograft cells, female LEW rats were irradiated and substituted with bone marrow from male LEW rats. Subsequently, the animals received an aortic transplant from female F344 rats or were exposed to a balloon injury of the carotid artery. Immunostaining and real-time polymerase chain reaction confirmed the above findings, but the fractions of leukocytes and SRY-positive cells were lower in the carotids than in the allografts. Combined primed in situ labeling and immunostaining verified that not only inflammatory but also smooth muscle-like cells of male origin appeared in the vessel wall in both situations. These observations suggest that the smooth-muscle cells that participate in the development of neointimal lesions during vascular disease may, in part, originate from circulating progenitors.

Anti-VEGF agents confer survival advantages to tumor-bearing mice by improving cancer-associated systemic syndrome.

The underlying mechanism by which anti-VEGF agents prolong cancer patient survival is poorly understood. We show that in a mouse tumor model, VEGF systemically impairs functions of multiple organs including those in the hematopoietic and endocrine systems, leading to early death. Anti-VEGF antibody, bevacizumab, and anti-VEGF receptor 2 (VEGFR-2), but not anti-VEGFR-1, reversed VEGF-induced cancer-associated systemic syndrome (CASS) and prevented death in tumor-bearing mice. Surprisingly, VEGFR2 blockage improved survival by rescuing mice from CASS without significantly compromising tumor growth, suggesting that “off-tumor” VEGF targets are more sensitive than the tumor vasculature to anti-VEGF drugs. Similarly, VEGF-induced CASS occurred in a spontaneous breast cancer mouse model overexpressing neu. Clinically, VEGF expression and CASS severity positively correlated in various human cancers. These findings define novel therapeutic targets of anti-VEGF agents and provide mechanistic insights into the action of this new class of clinically available anti-VEGF cancer drugs.

VEGF significantly restores impaired memory behavior in Alzheimer's mice by improvement of vascular survival

The functional impact of amyloid peptides (Aβs) on the vascular system is less understood despite these pathologic peptides are substantially deposited in the brain vasculature of Alzheimers patients. Here we show substantial accumulation of Aβs 40 and 42 in the brain arterioles of Alzheimers patients and of transgenic Alzheimers mice. Purified Aβs 1–40 and 1–42 exhibited vascular regression activity in the in vivo animal models and vessel density was reversely correlated with numbers and sizes of amyloid plaques in human patients. A significant high number of vascular cells underwent cellular apoptosis in the brain vasculature of Alzheimers patients. VEGF significantly prevented Aβ-induced endothelial apoptosis in vitro. Neuronal expression of VEGF in transgenic mice restored memory behavior of Alzheimers. These findings provide conceptual implication of improvement of vascular functions as a novel therapeutic approach for the treatment of Alzheimers disease.

Human Cytomegalovirus–Platelet Interaction Triggers Toll-Like Receptor 2–Dependent Proinflammatory and Proangiogenic Responses

Objective—Human cytomegalovirus (HCMV) is a widespread pathogen that correlates with various clinical complications, including atherosclerosis. HCMV is released into the circulation during primary infection and periodic viral reactivation, allowing virus–platelet interactions. Platelets are important in the onset and development of atherosclerosis, but the consequences of platelet–HCMV interactions are unclear. Approach and Results—We studied the effects of HCMV–platelet interactions in blood from healthy donors using the purified clinical HCMV isolate VR1814. We demonstrated that HCMV bound to a Toll-like receptor (TLR) 2–positive platelet subpopulation, which resulted in signal transduction, degranulation, and release of proinflammatory CD40L and interleukin-1&bgr; and proangiogenic vascular endothelial–derived growth factor. In mice, murine CMV activated wild-type but not TLR2-deficient platelets. However, supernatant from murine CMV–stimulated wild-type platelets also activated TLR2-deficient platelets, indicating that activated platelets generated soluble mediators that triggered further platelet activation, independent of TLR2 expression. Inhibitor studies, using ADP receptor antagonists and apyrase, revealed that ADP release is important to trigger secondary platelet activation in response to HCMV. HCMV-activated platelets rapidly bound to and activated neutrophils, supporting their adhesion and transmigration through endothelial monolayers. In an in vivo model, murine CMV induced systemic upregulation of platelet–leukocyte aggregates and plasma vascular endothelial–derived growth factor in mice and showed a tendency to enhance neutrophil extravasation in a TLR2-dependent fashion. Conclusions—HCMV is a well-adapted pathogen that does not induce immediate thrombotic events. However, HCMV–platelet interactions lead to proinflammatory and proangiogenic responses, which exacerbate tissue damage and contribute to atherogenesis. Therefore, platelets might contribute to the effects of HCMV in accelerating atherosclerosis.

Monocyte Chemoattractant Protein 1–Mediated Migration of Mesenchymal Stem Cells Is a Source of Intimal Hyperplasia

Objective—Intimal hyperplasia is considered to be a healing response and is a major cause of vessel narrowing after injury, where migration of vascular progenitor cells contributes to pathological events, including transplant arteriosclerosis. Approach and Results—In this study, we used a rat aortic–allograft model to identify the predominant cell types associated with transplant arteriosclerosis and to identify factors important in their recruitment into the graft. Transplantation of labeled adventitial tissues allowed us to identify the adventitia as a major source of cells migrating to the intima. RNA microarrays revealed a potential role for monocyte chemoattractant protein 1 (MCP-1), stromal cell–derived factor 1, regulated on activation, normal T cell expressed and secreted, and interferon-inducible protein 10 in the induced vasculopathy. MCP-1 induced migration of adventitial fibroblast cells. CCR2, the receptor for MCP-1, was coexpressed with CD90, CD44, NG2, or sca-1 on mesenchymal stem cells. In vivo experiments using MCP-1–deficient and CCR2-deficient mice confirmed an important role of MCP-1 in the formation of intimal hyperplasia in a mouse model of vascular injury. Conclusions—The adventitia is a potentially important cellular source that contributes to intimal hyperplasia, and MCP-1 is a potent chemokine for the recruitment of adventitial vascular progenitor cells to intimal lesions.

Pericyte–fibroblast transition promotes tumor growth and metastasis

Significance We show that vascular pericytes significantly contribute to cancer invasion and metastasis by the mechanism of the pericyte–fibroblast transition (PFT). This study proposes this concept and indicates the vascular pericyte’s role. Vascular pericytes were considered to remodel tumor vessels toward a mature phenotype. However, once dissociated from tumor vessels their functions within the tumor tissue are not known. In the present study, we show that pericytes, once detached from tumor microvasculatures, underwent differentiation to become stromal fibroblasts, which are known to contribute to tumor invasion and metastasis. Our results show that vascular pericytes are the important source of stromal fibroblasts and targeting PFT may offer a new treatment option in cancer metastasis. Vascular pericytes, an important cellular component in the tumor microenvironment, are often associated with tumor vasculatures, and their functions in cancer invasion and metastasis are poorly understood. Here we show that PDGF-BB induces pericyte–fibroblast transition (PFT), which significantly contributes to tumor invasion and metastasis. Gain- and loss-of-function experiments demonstrate that PDGF-BB-PDGFRβ signaling promotes PFT both in vitro and in in vivo tumors. Genome-wide expression analysis indicates that PDGF-BB–activated pericytes acquire mesenchymal progenitor features. Pharmacological inhibition and genetic deletion of PDGFRβ ablate the PDGF-BB–induced PFT. Genetic tracing of pericytes with two independent mouse strains, TN-AP-CreERT2:R26R-tdTomato and NG2-CreERT2:R26R-tdTomato, shows that PFT cells gain stromal fibroblast and myofibroblast markers in tumors. Importantly, coimplantation of PFT cells with less-invasive tumor cells in mice markedly promotes tumor dissemination and invasion, leading to an increased number of circulating tumor cells and metastasis. Our findings reveal a mechanism of vascular pericytes in PDGF-BB–promoted cancer invasion and metastasis by inducing PFT, and thus targeting PFT may offer a new treatment option of cancer metastasis.

High Prevalence of Human Cytomegalovirus in Brain Metastases of Patients with Primary Breast and Colorectal Cancers

BACKGROUND: Brain metastases (BMs) develop by largely unknown mechanisms and cause major morbidity and mortality in patients with solid tumors. Human cytomegalovirus (HCMV) is frequently detected in tumor tissue from patients with different cancers. Here, we aimed to determine the prevalence and potential prognostic role of HCMV in BMs. METHODS: We obtained archived samples of BMs from 41 patients with breast cancer and 37 with colorectal cancer and paired primary tumor tissues from 13 and 12 patients in each respective group. In addition, primary breast cancer tissues from 15 patients were included. HCMV proteins were detected with an immunohistochemical technique and Western blot. HCMV nucleic acids were detected with TaqMan polymerase chain reaction (PCR) assay. RESULTS: HCMV proteins were abundantly expressed in 99% of BM specimens, and in 12 of 13 (92%) paired primary breast cancer specimens. All 12 paired colon cancer samples were positive for HCMV proteins. Protein staining was mainly confined to neoplastic cells. Western blot analysis detected an HCMV-IE reactive protein in 53% of breast cancer specimens, and PCR detected the presence of HCMV DNA and transcripts in 92% and 80% of samples, respectively. Patients with high-level expression of HCMV-IE proteins in their tumors had a shorter time to tumor progression and shorter overall survival. CONCLUSIONS: The prevalence of HCMV proteins and nucleic acids is very high in primary and metastatic tumors and may drive the development of metastatic brain tumors; therefore, this virus may represent a potential therapeutic target in metastatic cancer.

Inhibition of rat smooth muscle cell adhesion and proliferation by non-anticoagulant heparins.

Heparin is a well established growth inhibitor of arterial smooth muscle cells (SMCs) both in animal models and in vitro. Even though the cellular mechanisms involved in the anti‐proliferative properties of heparin are being resolved, the structural requirements for the biological effects of heparin are not known in detail. Here, we have studied the effect of chemically modified heparins of different molecular weights and anticoagulant activities on proliferation and adhesion of rat aortic SMCs in vitro. The effects of native heparin (NH) and chemically modified heparins were examined after stimulation with fetal calf serum (FCS), platelet‐derived growth factor BB (PDGF BB), basic fibroblast growth factor (bFGF), and heparin‐binding epidermal growth factor (hbEGF) with respect to DNA synthesis and expression of phosphorylated and activated mitogen‐activated protein kinase (pERK1 and 2). In a similar manner as NH, the modified heparins were capable of inhibiting activation of ERK1 and 2 and DNA synthesis induced by FCS and hbEGF whereas the modified heparins potentiated the mitogenic effect of bFGF and no compound affected PDGF BB‐induced ERK activity and SMC growth. In contrast, cell adhesion to fibronectin was inhibited by NH and modified heparins in a size‐dependent manner with the lowest effect by the smallest compound. The results show that heparins with varying anticoagulant activities and molecular weights but with similar sulfate content can retain anti‐proliferative properties while the effect on some other biological processes such as cell adhesion is lost. Possibly, such chemical alterations may yield useful substances for the prevention of SMC proliferation after arterial injury. J. Cell. Physiol. 193: 365–372, 2002.