Invasion Of Microvascular Endothelial Cells Research Articles

<i>Streptococcus suis</i> Type 2 SSU0587 Protein is a Beta-Galactosidase That Contributes to Bacterial Adhesion but Not to Virulence in Mice

ABSTRACTBacterial surface proteins play key roles in virulence and often contribute to bacterial adhesion and invasion. We discovered that the Streptococcus suis type 2 (SS2) gene SSU0587 encodes a protein of 1,491 amino acids that possesses β-galactosidase activity. The surface association of the protein was dependent upon sortase activity. Deleting SSU0587 from clinical SS2 isolate JX081101 caused a loss of both β-galactosidase activity and adherence to microvascular endothelial cells. Deleting SSU0587 had no measurable impact on either invasion of microvascular endothelial cells or on virulence in a murine infection model, although the concentration of JX081101ΔSSU0587 was reduced in the brains of infected mice, as compared with the pathogen loads of the wild-type strain.

Abstract 361: ZNF24 may modulate microvascular endothelial cell invasion by regulating MMP2 transcription.

Abstract ZNF24 is a C2H2 zinc finger transcription factor that regulates angiogenesis by repressing the transcription of vascular endothelial growth factor (VEGF). It has also been implicated in the control of cell proliferation and differentiation in the central nervous system and in hepatocellular carcinoma. In the current study, we have focused on the role of ZNF24 in modulating endothelial cell motility. We have found that silencing of ZNF24 in human microvascular endothelial cells (HMVEC) leads to significantly decreased cell migration and invasion. Consistently, the protein level and proteolytic activity of matrix metalloproteinase-2 (MMP2), but not those of matrix metalloproteinase-9 (MMP9), are significantly downregulated when ZNF24 is silenced in these cells. We have also determined the effect of ZNF24 silencing on the mRNA levels of endogenous inhibitors of MMPs, the tissue inhibitors of metalloproteinases (TIMPs). While TIMP1 and TIMP4 levels are moderately upregulated by ZNF24 silencing, the level of TIMP2, a major regulator of MMP2 proteolytic activity, and that of TIMP3, are significantly decreased. However, ZNF24 siRNA induces only a moderate decrease in the protein level of TIMP2, suggesting that TIMP2 does not play an important role in the regulation of MMP9 activity by ZNF24. Another major regulator of MMP2 proteolytic activity, membrane-type-1 matrix metalloproteinase (MT1-MMP), also does not exhibit significant changes when ZNF24 is silenced, suggesting that MMP2 is regulated by ZNF24 at the transcriptional level. Indeed, the mRNA level of MMP2 is significantly downregulated when cells are transfected with ZNF24 siRNA, indicating that ZNF24 positively regulates the transcription of MMP2. When compared to normal endothelial cells, tumor endothelial cells exhibit higher levels of endogenous ZNF24, suggesting that ZNF24 may function to increase cell invasion in these cells via upregulation of MMP2. Our current studies are focused on determining whether MMP2 is a direct downstream target of ZNF24 as well as on its role(s) in modulating endothelial cell motility in vivo. (Supported by NIH P01 CA045548, the Breast Cancer Research Foundation and the Advanced Medical Research Foundation) Citation Format: Di Jia, Marsha A. Moses. ZNF24 may modulate microvascular endothelial cell invasion by regulating MMP2 transcription. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 361. doi:10.1158/1538-7445.AM2013-361

Induction of Brain Microvascular Endothelial Cell Urokinase Expression by Cryptococcus neoformans Facilitates Blood-Brain Barrier Invasion

The invasive ability of the blood-borne fungal pathogen Cryptococcus neoformans can be enhanced through interactions with host plasma components, such as plasminogen. Previously we showed by in vitro studies that plasminogen coats the surface of C. neoformans and is converted to the active serine protease, plasmin, by host plasminogen activators. Viable, but not formaldehyde- or sodium azide-killed, cryptococcal strains undergo brain microvascular endothelial cell-dependent plasminogen-to-plasmin activation, which results in enhanced, plasmin-dependent cryptococcal invasion of primary bovine brain microvascular endothelial cells and fungal ability to degrade plasmin substrates. In the present work, brain microvascular endothelial cells cultured with viable, but not killed, cryptococcal strains led to significant increases in both urokinase mRNA transcription and cell-associated urokinase protein expression. Soluble urokinase was also detected in conditioned medium from brain microvascular endothelial cells cultured with viable, but not killed, C. neoformans. Exposure of plasminogen pre-coated viable C. neoformans to conditioned medium from strain-matched brain microvascular endothelial cell-fungal co-cultures resulted in plasminogen-to-plasmin activation and plasmin-dependent cryptococcal invasion. siRNA-mediated silencing of urokinase gene expression or the use of specific inhibitors of urokinase activity abrogated both plasminogen-to-plasmin activation on C. neoformans and cryptococcal-brain microvascular endothelial cell invasion. Our results suggest that pathogen exploitation of the host urokinase-plasmin(ogen) system may contribute to C. neoformans virulence during invasive cryptococcosis.

Involvement of PKCα–MAPK/ERK-phospholipase A2 pathway in the Escherichia coli invasion of brain microvascular endothelial cells

Escherichia coli K1 is the most common Gram-negative organism that causes neonatal meningitis following penetration of the blood–brain barrier. In the present study we demonstrated the involvement of cytosolic (cPLA2) and calcium-independent phospholipase A2 (iPLA2) and the contribution of cyclooxygenase-2 products in E. coli invasion of microvascular endothelial cells. The traversal of bacteria did not determine trans-endothelial electrical resistance (TEER) and ZO-1 expression changes and was reduced by PLA2s siRNA. cPLA2 and iPLA2 enzyme activities and cPLA2 phosphorylation were stimulated after E. coli incubation and were attenuated by PLA2, PI3-K, ERK 1/2 inhibitors. Our results demonstrate the role of PKCα/ERK/MAPK signaling pathways in governing the E. coli penetration into the brain.

Mycoplasmal cerebral vasculopathy in a lymphoma patient: Presumptive evidence of Mycoplasma pneumoniae microvascular endothelial cell invasion in a brain biopsy

A 73-year-old man had episodic encephalopathy, ataxia and neuropathy. Symptoms largely resolved but adenopathy later lead to the diagnosis of a low-grade follicular lymphoma. The neurological symptoms soon recurred with new pontine calcifications identified by computed tomography. Brain biopsy revealed microvascular endothelial cell nuclear changes. Electron microscopy identified small polymorphic bacteria without a cell wall and with terminal and attachment organelles within endothelial cells and clustered in some microvascular lumina. Immunostaining was positive for Mycoplasma pneumoniae and convalescent serum enzyme immunoassay was positive for M. pneumoniae IgG. The patient again recovered and he was neurologically stable 33 months after the initial episode. The ultrastructural findings of the bacterial cells are distinctive of some mycoplasmal species when compared to other small bacteria. Mycoplasma-like organisms are reported in four autopsied patients who had chronic encephalopathy, movement disorders, and some of the same light- and electron-microscopic findings in the brain as our patient. Direct neuroinvasion by Mycoplasma species has been suggested, while anatomic observations in our patient and in the four autopsy cases show microvascular invasion but not parenchymal invasion. Most mycoplasmal encephalitis may be immune-mediated. The frequency of neurovascular invasion is not known. It may be rare and it may persist.

α5β1 Integrin Ligand PHSRN Induces Invasion and α5 mRNA in Endothelial Cells to Stimulate Angiogenesis

Angiogenesis requires endothelial cell invasion and is crucial for wound healing and for tumor growth and metastasis. Invasion of native collagen is mediated by the α5β1 integrin fibronectin receptor. Thus, α5β1 up-regulation on the surfaces of endothelial cells may induce endothelial cell invasion to stimulate angiogenesis. We report that the interaction of α5β1 with its PHSRN peptide ligand induces human microvascular endothelial cell invasion and that PHSRN-induced endothelial cell invasion is regulated by α4β1 integrin and requires matrix metalloproteinase 1 (MMP-1). Moreover, our results show that exposure to PHSRN causes rapid, specific up-regulation of surface levels of α5β1 integrin and significantly increases α5 integrin mRNA in microvascular endothelial cells. Consistent with these results, α5 small interfering RNA abrogates PHSRN-induced surface α5 and MMP-1 up-regulation, as well as blocking invasion induction. We also observed dose-dependent, PHSRN-induced α5β1 integrin up-regulation on endothelial cells in vivo in Matrigel plugs. We further report that the PHSCN peptide, an α5β1-targeted invasion inhibitor, blocks PHSRN-induced invasion, α5β1 up-regulation, α5 mRNA induction, and MMP-1 secretion in microvascular endothelial cells and that systemic PHSCN administration prevents PHSRN-induced α5β1 up-regulation and angiogenesis in Matrigel plugs. These results demonstrate a critical role for α5β1 integrin and MMP-1 in mediating the endothelial cell invasion and angiogenesis and suggest that PHSRN-induced α5 transcription and α5β1 up-regulation may form an important feed-forward mechanism for stimulating angiogenesis.

Ca2+/calmodulin-dependent invasion of microvascular endothelial cells of human brain by Escherichia coli K1

Escherichia coli K1 invasion of microvascular endothelial cells of human brain (HBMEC) is required for E. coli penetration into the central nervous system, but the microbial-host interactions that are involved in this invasion of HBMEC remain incompletely understood. We have previously shown that FimH, one of the E. coli determinants contributing to the binding to and invasion of HBMEC, induces Ca(2+) changes in HBMEC. In the present study, we have investigated in detail the role of cellular calcium signaling in the E. coli K1 invasion of HBMEC, the main constituents of the blood-brain barrier. Addition of the meningitis-causing E. coli K1 strain RS218 (O18:K1) to HBMEC results in transient increases of intracellular free Ca(2+). Inhibition of phospholipase C with U-73122 and the chelating of intracellular Ca(2+) by BAPTA/AM reduces bacterial invasion of HBMEC by approximately 50%. Blocking of transmembrane Ca(2+) fluxes by extracellular lanthanum ions also inhibits the E. coli invasion of HBMEC by approximately 50%. In addition, E. coli K1 invasion is significantly inhibited when HBMEC are pretreated by the calmodulin antagonists, trifluoperazine or calmidazolium, or by ML-7, a specific inhibitor of Ca(2+)/calmodulin-dependent myosin light-chain kinase. These findings indicate that host intracellular Ca(2+) signaling contributes in part to E. coli K1 invasion of HBMEC.

Identification of Escherichia coli K1 genes contributing to human brain microvascular endothelial cell invasion by differential fluorescence induction.

Most cases of Escherichia coli K1 meningitis arise as a result of haematogenous spread, however there is a limited understanding of the mechanisms by which circulating E. coli K1 cross the blood-brain barrier. We have previously shown that environmental growth conditions both positively and negatively influence the capabilities of E. coli K1 to invade brain microvascular endothelial cells (BMEC), for example growth in media supplemented with 50% newborn bovine serum (NBS) increased BMEC invasion, whereas growth in media supplemented with 0.2 M NaCl repressed invasion in vitro and in vivo. In this study, differential fluorescence induction (DFI) was used to identify E. coli K1 genes involved in this differentially expressed invasion phenotype. E. coli K1 promoter libraries were constructed and screened for gfp expression in a manner analogous to the above growth conditions. Twenty-four clones were isolated that showed fluorescence induction when grown under the invasion-enhancing condition (i.e. NBS). Four of these clones also demonstrated repression or no induction of fluorescence when grown under the invasion-repressing condition (i.e. 0.2 M NaCl). One such clone, containing a ygdP promoter and an open reading frame (ORF), showed significant homology to Bartonella bacilliformis IalA (invasion associated locus). Among the other NBS-inducing loci, finPtraJ was identified as well as several clones with no homology to other known genes. When ygdP, finPtraJ and several of the unique loci were disrupted in E. coli K1, there was a significant decrease in human BMEC (HBMEC) invasion. RNA transcript analysis determined that these newly identified invasion loci were differentially regulated at the transcriptional level. This is the first demonstration of using DFI to identify E. coli K1 genes contributing to HBMEC invasion.

CD44-related chondroitin sulfate proteoglycan, a cell surface receptor implicated with tumor cell invasion, mediates endothelial cell migration on fibrinogen and invasion into a fibrin matrix.

Microvascular endothelial cell invasion into the fibrin provisional matrix is an integral component of angiogenesis during wound repair. Cell surface receptors which interact with extracellular matrix proteins participate in cell migration and invasion. Malignant cells use CD44-related chondroitin sulfate proteoglycan (CSPG) as a matrix receptor to mediate migration and invasion. In this study, we examine whether cell surface CSPG can mediate similar events in nonmalignant wound microvascular endothelial cells or whether use of CSPG for migration and invasion is a property largely restricted to malignant cells. After inhibiting CSPG synthesis with p-nitrophenyl beta-d xylopyranoside (beta-d xyloside), wound microvascular endothelial cells were capable of attaching and spreading on the surface of a fibrin gel; however, their ability to invade the fibrin matrix was virtually eliminated. To begin to examine the mechanism by which endothelial cells use CSPG to invade fibrin matrices, cell adhesion and migration on fibrinogen was examined. Endothelial cell adhesion and migration on fibrinogen were inhibited by both beta-d xyloside and after cleavage of chondroitin sulfate from the core protein by chondroitinase ABC. We have determined that wound microvascular endothelial cells express the majority of their proteoglycan as CSPG and that the CSPG core protein is immunologically related to CD44. PCR studies show that these cells express both the "standard" (CD44H) isoform and an isoform containing the variably spliced exon V3. In addition, anti-CD44 antibody blocks endothelial cell migration on fibrinogen. Affinity chromatography studies reveal that partially purified microvascular endothelial cell CSPG binds fibrinogen. These findings suggest that CD44-related CSPG, a molecule implicated in the invasive behavior of tumor cells, is capable of binding fibrinogen/fibrin, thereby mediating endothelial cell migration and invasion into the fibrin provisional matrix during wound repair.