Rapid loss of microvascular integrin expression during focal brain ischemia reflects neuron injury.

The integrity of cerebral microvessels requires the close apposition of the endothelium to the astrocyte endfeet. Integrins alpha1beta1 and alpha6beta4 are cellular matrix receptors that may contribute to cerebral microvascular integrity. It has been hypothesized that focal ischemia alters integrin expression in a characteristic time-dependent manner consistent with neuron injury. The effects of middle cerebral artery occlusion (MCAO) and various periods of reperfusion on microvasclar integrin alpha1beta1 and alpha6beta4 expression were examined in the basal ganglia of 17 primates. Integrin subunits alpha1 and beta1 colocalized with the endothelial cell antigen CD31 in nonischemic microvessels and with glial fibrillary acidic protein on astrocyte fibers. Rapid, simultaneous, and significant disappearance of both integrin alpha1 and beta1 subunits and integrin alpha6beta4 occurred by 2 hours MCAO, which was greatest in the region of neuron injury (ischemic core, Ic), and progressively less in the peripheral (Ip) and nonischemic regions (N). Transcription of subunit beta1 mRNA on microvessels increased significantly in the Ic/Ip border and in multiple circular subregions within Ic. Microvascular integrin alpha1beta1 and integrin alpha6beta4 expression are rapidly and coordinately lost in Ic after MCAO. With loss of integrin alpha1beta1, multiple regions of microvascular beta1 mRNA up-regulation within Ic suggest that microvessel responses to focal ischemia are dynamic, and that multiple cores, not a single core, are generated. These changes imply that microvascular integrity is modified in a heterogeneous, but ordered pattern.

Presenilin-1 and -2 are molecular targets for gamma-secretase inhibitors.

Presenilins are integral membrane protein involved in the production of amyloid beta-protein. Mutations of the presenilin-1 and -2 gene are associated with familial Alzheimer's disease and are thought to alter gamma-secretase cleavage of the beta-amyloid precursor protein, leading to increased production of longer and more amyloidogenic forms of A beta, the 4-kDa beta-peptide. Here, we show that radiolabeled gamma-secretase inhibitors bind to mammalian cell membranes, and a benzophenone analog specifically photocross-links three major membrane polypeptides. A positive correlation is observed among these compounds for inhibition of cellular A beta formation, inhibition of membrane binding and cross-linking. Immunological techniques establish N- and C-terminal fragments of presenilin-1 as specifically cross-linked polypeptides. Furthermore, binding of gamma-secretase inhibitors to embryonic membranes derived from presenilin-1 knockout embryos is reduced in a gene dose-dependent manner. In addition, C-terminal fragments of presenilin-2 are specifically cross-linked. Taken together, these results indicate that potent and selective gamma-secretase inhibitors block A beta formation by binding to presenilin-1 and -2.

Regulation of plasminogen gene expression by interleukin-6.

Plasmin, the primary fibrinolytic enzyme, has a broad substrate spectrum and participates in other biological processes dependent upon proteolytic activity. Consequently, plasmin activity is tightly regulated by plasminogen activators and protease inhibitors. In this study, we examined whether regulation of plasminogen gene expression also might provide a new mechanism for controlling this system. We examined the effects of recombinant human interleukin-6 (rhIL-6), a pleiotropic cytokine, on plasminogen mRNA expression in primary murine hepatocytes and Hep3B human hepatoma cells. In primary hepatocytes, rhIL-6 and hydrocortisone separately increased plasminogen mRNA expression, but hydrocortisone did not markedly enhance the response to rhIL-6. Hep3B hepatoma cells exhibited more modest responses to rhIL-6. We used the polymerase chain reaction to amplify a 1,067-bp fragment of the human plasminogen promoter/5' flanking region. This fragment was cloned upstream of a luciferase reporter gene. Hep3B cells transiently transfected with this construct provided approximately 100-fold higher luciferase activity compared to cells transfected with control plasmids, and luciferase activity was increased approximately 4.5-fold when these cells were treated with rhIL-6. Furthermore, mice injected with rhIL-6 exhibited increases in hepatic plasminogen mRNA. Circulating plasminogen levels were significantly higher in the mice injected with rhIL-6 compared to mice injected with saline. Mice injected with lipopolysaccharide (an inducer of IL-6 in vivo) also showed increased hepatic plasminogen mRNA. Thus, plasminogen gene expression can be modulated by rhIL-6, suggesting a new mechanism for regulating biological systems that use plasmin.

Type 1 plasminogen activator inhibitor induces multimerization of plasma vitronectin. A suggested mechanism for the generation of the tissue form of vitronectin in vivo.

The conformation and degree of multimerization of vitronectin (Vn) appears to be of critical importance for its functions, but little is known about the underlying mechanisms that control Vn multimerization. We report that Vn secreted by cultured hepatoma cells is present as a mixture of monomeric and multimeric forms. A single protein of Mr 45,000 co-purified with hepatoma cell-derived Vn, which was immunologically identified as type 1 plasminogen activator inhibitor (PAI-1). The possibility that PAI-1 may modulate Vn multimerization was investigated. The addition of active PAI-1 to unfractionated plasma containing Vn monomers resulted in the formation of covalently and noncovalently associated Vn multimers and expression of conformationally sensitive epitopes. In contrast, inactive forms of PAI-1 did not efficiently induce Vn multimerization and conformational change. Gel filtration analysis revealed that Vn remained multimeric after dissociation from PAI-1. Vn multimers were also assembled using purified monomeric Vn and PAI-1, suggesting that a plasma cofactor was not required to induce Vn multimerization. This study provides insights into physiological mechanism responsible for the generation of homomultimeric Vn, a multimeric form of Vn that is not in complex with other proteins and which expresses a functional repertoire distinct from that of plasma Vn.

Characterization of the denaturation and renaturation of human plasma vitronectin. II. Investigation into the mechanism of formation of multimers.

Unfolding and refolding of plasma vitronectin appear irreversible under near physiological conditions, with rearrangements of disulfides and self-association to a multimeric form observed as prominent structural alterations which accompany denaturation. A mechanism for the folding reactions of vitronectin has been proposed (Zhuang, P., Blackburn, M. N., and Peterson, C. B.(1996) J. Biol. Chem. 270, 14323-14332) in which vitronectin acquires a partially folded intermediate structure which is highly prone to oligomerize into a multimeric form. Strongly oxidizing conditions adopted for refolding from urea were effective at preventing disulfide rearrangement which disrupts distal disulfides near the C terminus of the protein. Prohibiting disulfide rearrangement under these conditions, however, was not sufficient to achieve reversibility in folding. In contrast, variations in the ionic strength of the refolding medium affect the partitioning of species so that refolded monomers are obtained at high ionic strength, and self-association is precluded. The effects of ionic strength on the partially folded intermediate in the vitronectin folding pathway appear to favor intramolecular hydrophobic collapse to form a stable hydrophobic core for the monomer versus intermolecular hydrophobic interactions which stabilize multimeric vitronectin. Although both ionic and hydrophobic interactions presumably contribute to subunit interfaces within the multimer, the basic heparin-binding region near the C terminus of the protein does not provide binding interactions which are important for self-association of vitronectin.

Hydrolysis of platelet vitronectin by calpain.

Vitronectin (Vn) is not only a major adhesive glycoprotein present in platelets but also regulates proteolytic enzyme cascades, including the blood coagulation, fibrinolytic, and complement systems. In human platelet lysates prepared by freeze-thawing or by the addition of nonionic detergent, the Vn antigen content was drastically reduced in comparison with lysates prepared in the presence of SDS, suggesting that Vn is hydrolyzed by platelet-associated enzymes. Exogenously added purified human Vn and Vn present in plasma were also cleaved by these enzyme systems. Degradation was mediated by a nonsecreted or membrane-associated protease system that was inhibited by E-64, EDTA, and leupeptin but not inhibitors of serine and aspartic proteases, suggesting an involvement of calcium-dependent cysteine proteases. Consistently, calpastatin inhibited the hydrolysis of Vn, suggesting that Vn is a substrate for calpain. This was confirmed in a purified system. Vn was cleaved by calpains I and II in a dose- and time-dependent manner, resulting in defined Vn fragments with similar electrophoretic mobility in comparison with those detected in platelet lysates. Functional characterization of the calpain-hydrolyzed Vn revealed that while the type 1 plasminogen activator inhibitor binding activity was unchanged, the heparin and cell binding functions were destroyed. These results suggest that calpains released upon platelet membrane damage or upon tissue injury and necrosis differentially regulate functional domains of the Vn molecule.

Differential regulation of vitronectin in mice and humans in vitro.

To define the cis-acting elements involved in the regulation of the murine vitronectin (Vn) gene in inflammation, the 5'-flanking region was isolated, fused to the luciferase reporter gene, and the basal and interleukin 6 (IL-6)-stimulated transcriptional activity was tested in transfection experiments using Hep3B cells. Treatment with IL-6 induced this construct by more than 20-fold, whereas the corresponding 5'-flanking region of the human Vn gene was not stimulated. Transfection studies using murine Vn constructs with serial 5'-deletions revealed that two sequences were important in the IL-6 response, and specific mutations in both sequences abolished the response. A 2-base pair mutation converted the human sequence to that of a murine IL-6 responsive element and partially conveyed IL-6 inducibility. In contrast, transforming growth factor beta stimulated the human construct and the endogenous Vn gene in human Hep3B cells in a dose-dependent manner, whereas the murine construct was not responsive. The transforming growth factor beta responsive region was localized to a 30-base pair fragment with little homology to the murine sequence. These studies reveal that the structural basis for the differential regulation of the human and murine Vn genes resides in the differences in promoter sequence.

Detection of vitronectin in mineralized bone matrix.

Adhesive glycoproteins in the bone matrix are of critical importance for cell anchorage, proliferation, migration, differentiation, and regulation of bone metabolism. The localization of the adhesive glycoprotein vitronectin (Vn) in murine bone tissue was evaluated by immunohistochemical staining. Vitronectin was present throughout the mineralized bone matrix of cancellous and cortical bone, whereas cartilage was devoid of Vn staining. To exclude the possibility that the positive Vn staining resulted from plasma Vn in blood vessels within the bone sections, adjacent tissue sections were stained with antibodies to fibrinogen, and abundant plasma protein. Fibrinogen immunoreactivity was confined to blood vessels in the bone marrow and Haversian system, whereas the mineralized bone matrix was devoid of staining. The presence of Vn in murine bones was confirmed by sequential extraction, followed by fractionation of the resulting polypeptides by gel electrophoresis and immunoblotting analysis. Hydroxyapatite affinity chromatography raises the possibility that mineral interactions, at least in part, mediate the incorporation of Vn into the bone matrix. These results indicate that Vn is a specific component of bone tissue and raise the possibility that Vn is involved in regulation of bone metabolism.

IL-6 stimulates vitronectin gene expression in vivo.

We tested the hypothesis that vitronectin (Vn) is regulated as an acute phase reactant in response to inflammatory stimuli. In initial experiments, Vn levels were measured during the surgically induced acute phase response in humans. The plasma concentration of Vn increased approximately twofold following elective orthopedic surgery and remained elevated up to 5 days. To examine the mechanism(s) of increased Vn synthesis, hepatic Vn mRNA expression and serum levels were examined in three rat models of acute inflammation: LPS (i.v.), CFA (i.p.), or turpentine (s.c.) injection. The serum concentration of Vn increased approximately twofold 24 h following treatment with turpentine. The expression of Vn mRNA in the liver increased markedly as early as 3 h after treatment in these models and remained elevated up to 18 h. Northern blot analysis of RNA isolated from fractionated liver cells derived from rats treated with LPS indicated that Vn was mainly expressed in hepatocytes, but not in the endothelial or nonparenchymal cell fractions. To analyze the individual effects of raised corticosterone and IL-6 levels on the expression of hepatic Vn mRNA, rats were injected (i.p.) with either dexamethasone or purified recombinant rat IL-6. Vn mRNA expression was elevated within 1 h after IL-6 injection, whereas dexamethasone-injected rats showed unchanged Vn expression. Vn mRNA also was increased in rats chronically injected with IL-6. These results indicate that the Vn gene is up-regulated in acute and chronic inflammation, and this induction is primarily mediated by IL-6.

The somatomedin B domain of vitronectin. Structural requirements for the binding and stabilization of active type 1 plasminogen activator inhibitor.

We recently localized the high affinity binding site for activated type 1 plasminogen activator inhibitor (PAI-1) to the somatomedin B domain (i.e. amino acid (aa) 1-51) of vitronectin (Vn). In this study to further define this site, N-terminal Vn fragments of various lengths were expressed in Escherichia coli and tested for PAI-1 binding activity. Vn polypeptides containing aa 1-52 and 1-40 retained PAI-1 binding activity and stabilized PAI-1 to a similar extent as intact Vn, but polypeptides containing aa 1-30 did not bind to PAI-1 nor stabilize its activity. The effects of monoclonal antibodies (mAbs) to Vn on PAI-1 binding was also determined. One mAb bound to Vn and blocked its ability to bind to PAI-1. It also dissociated pre-existing PAI-1.Vn complexes, and prevented the incorporation of PAI-1 into extracellular matrix of HT 1080 cells. This mAb bound to the recombinant peptide containing aa 1-40, but not to the peptide consisting of aa 1-30. A second randomly chosen mAb with similar affinity for Vn was inactive in these assays and bound to the region between aa 52 and 239. These results indicate that the high affinity binding site for active PAI-1 in Vn is between aa 1 and 40, and that this domain may also stabilize active PAI-1.

Interactions between type 1 plasminogen activator inhibitor, extracellular matrix and vitronectin.

Regulation of plasminogen activation is a key process in controlling proteolytic events in the extracellular matrix (ECM) and this regulation is achieved through the action of specific plasminogen activator (PA) inhibitors (PAIs). Type I PAI (PAI-1) is the physiological inhibitor both of urinary-type PA (u-PA) and tissue-type PA (t-PA) (Loskutoff et al., 1989) and is a major component of the ECM of cultured cells. This inhibitor may protect ECM constituents against cellular proteases and thus influence the cell migration and tissue destruction that occurs during development, inflammation and tumor metastasis. In this review, we discuss the properties of PAI-1 and the evidence that the binding of PAI-1 to ECM is mediated by serum-derived vitronectin (Vn).

Serum-derived vitronectin influences the pericellular distribution of type 1 plasminogen activator inhibitor.

Bovine aortic endothelial cells (BAEs) were used as a model system to study the nature and origin of protein(s) in the extracellular matrix that bind to type 1 plasminogen activator inhibitor (PAI-1). Matrix samples were fractionated by SDS-PAGE and analyzed by PAI-1 ligand binding and by immunoblotting using antibodies to vitronectin (Vn). PAI-1 bound primarily to two Vn-related polypeptides of Mr 63,000 and 57,000, and both of these partially degraded polypeptides were present in the culture serum. Radiolabeling experiments failed to detect significant Vn biosynthesis by BAEs (less than 0.03% of total), or by human umbilical vein endothelial cells and HT 1080 cells. The binding of PAI-1 to Vn was relatively specific since direct binding studies failed to demonstrate significant interactions between PAI-1 and other matrix proteins (e.g., fibronectin, type IV collagen, laminin, or matrigel). Kinetic studies indicate that PAI-1 rapidly accumulates in the matrix when BAEs are plated on Vn, appearing in the conditioned medium only after a significant lag period (1-2 h). However, no PAI-1 was detected in the matrix when the cells were plated on fibronectin-coated dishes, and there was no lag period for PAI-1 accumulation in the medium. These results indicate that PAI-1 binds specifically to serum-derived Vn in the matrix, and suggest that the composition of both the matrix and serum itself may influence the pericellular distribution of this important inhibitor.