The key role of transient receptor potential melastatin-2 channels in amyloid-ß-induced neurovascular dysfunction.

Alzheimer's dementia is a devastating and incurable disease afflicting over 35 million people worldwide. Amyloid-ß (Aß), a key pathogenic factor in this disease, has potent cerebrovascular effects that contribute to brain dysfunction underlying dementia by limiting the delivery of oxygen and glucose to the working brain. However, the downstream pathways responsible for the vascular alterations remain unclear. Here we report that the cerebrovascular dysfunction induced by Aß is mediated by DNA damage caused by vascular oxidative-nitrosative stress in cerebral endothelial cells, which, in turn, activates the DNA repair enzyme poly(ADP)-ribose polymerase. The resulting increase in ADP ribose opens transient receptor potential melastatin-2 (TRPM2) channels in endothelial cells leading to intracellular Ca(2+) overload and endothelial dysfunction. The findings provide evidence for a previously unrecognized mechanism by which Aß impairs neurovascular regulation and suggest that TRPM2 channels are a potential therapeutic target to counteract cerebrovascular dysfunction in Alzheimer's dementia and related pathologies.

Angiotensin II type 2 receptors have a major somatodendritic distribution in vasopressin-containing neurons in the mouse hypothalamic paraventricular nucleus.

The hypothalamic paraventricular nucleus (PVN) and angiotensin II (AngII) play critical roles in cardiovascular and neurohumoral regulation ascribed in part to vasopressin (VP) release. The AngII actions in the PVN are mediated largely through angiotensin II type 1 (AT1) receptors. However, there is indirect evidence that the functionally elusive central angiotensin II type 2 (AT2) receptors are also mediators of AngII signaling in the PVN. We used electron microscopic dual immunolabeling of antisera recognizing the AT2 receptor and VP to test the hypothesis that mouse PVN neurons expressing VP are among the cellular sites where this receptor has a subcellular distribution conducive to local activation. Immunoreactivity for the AT2 receptor was detected in somatodendritic profiles, of which approximately 60% of the somata and approximately 28% of the dendrites also contained VP. In comparison with somata and dendrites, axons, axon terminals, and glia less frequently contained the AT2 receptor. Somatic labeling for the AT2 receptor was often seen in the cytoplasm near the Golgi lamellae and other endomembrane structures implicated in receptor trafficking. AT2 receptor immunoreactivity in dendrites was commonly localized to cytoplasmic endomembranes, but was occasionally observed on extra- or peri-synaptic portions of the plasma membrane apposed by astrocytic processes or by unlabeled axon terminals. The labeled dendritic plasmalemmal segments containing AT2 receptors received asymmetric excitatory-type or more rarely symmetric inhibitory-type contacts from unlabeled axon terminals containing dense core vesicles, many of which are known to store neuropeptides. These results provide the first ultrastructural evidence that AT2 receptors in PVN neurons expressing VP and other neuromodulators are strategically positioned for surface activation by AngII and/or intracellular trafficking.

Angiotensin II subtype 1A (AT1A) receptors in the rat sensory vagal complex: subcellular localization and association with endogenous angiotensin.

Angiotensin II (Ang II) type 1 (AT1) receptors are prevalent in the sensory vagal complex including the nucleus tractus solitarii (NTS) and area postrema, each of which has been implicated in the central cardiovascular effects produced by Ang II. In rodents, these actions prominently involve the AT1A receptor. Thus, we examined the electron microscopic dual immunolabeling of antisera recognizing the AT1A receptor and Ang II to determine interactive sites in the sensory vagal complex of rat brain. In both the area postrema and adjacent dorsomedial NTS, many somatodendritic profiles were dually labeled for the AT1A receptor and Ang II. In these profiles, AT1A receptor-immunoreactivity was often seen in the cytoplasm beneath labeled portions of the plasma membrane and in endosome-like granules as well as Golgi lamellae and outer nuclear membranes. In addition, AT1A receptor labeling was detected on the plasma membrane and in association with cytoplasmic membranes in many small axons and axon terminals. These terminals were morphologically heterogeneous containing multiple types of vesicles and forming either inhibitory- or excitatory-type synapses. In the area postrema, AT1A receptor labeling also was detected in many non-neuronal cells including glia, capillary endothelial cells and perivascular fibroblasts that were less prevalent in the NTS. We conclude that in the rat sensory vagal complex, AT1A receptors are strategically positioned for involvement in modulation of the postsynaptic excitability and intracrine hormone-like effects of Ang II. In addition, these receptors have distributions consistent with diverse roles in regulation of transmitter release, regional blood flow and/or vascular permeability.

Carbon monoxide generated by heme oxygenase 1 suppresses endothelial cell apoptosis.

Heme oxygenase 1 (HO-1) inhibits apoptosis by regulating cellular prooxidant iron. We now show that there is an additional mechanism by which HO-1 inhibits apoptosis, namely by generating the gaseous molecule carbon monoxide (CO). Overexpression of HO-1, or induction of HO-1 expression by heme, protects endothelial cells (ECs) from apoptosis. When HO-1 enzymatic activity is blocked by tin protoporphyrin (SnPPIX) or the action of CO is inhibited by hemoglobin (Hb), HO-1 no longer prevents EC apoptosis while these reagents do not affect the antiapoptotic action of bcl-2. Exposure of ECs to exogenous CO, under inhibition of HO-1 activity by SnPPIX, substitutes HO-1 in preventing EC apoptosis. The mechanism of action of HO-1/CO is dependent on the activation of the p38 mitogen-activated protein kinase (MAPK) signaling transduction pathway. Expression of HO-1 or exposure of ECs to exogenous CO enhanced p38 MAPK activation by TNF-alpha. Specific inhibition of p38 MAPK activation by the pyridinyl imidazol SB203580 or through overexpression of a p38 MAPK dominant negative mutant abrogated the antiapoptotic effect of HO-1. Taken together, these data demonstrate that the antiapoptotic effect of HO-1 in ECs is mediated by CO and more specifically via the activation of p38 MAPK by CO.

TNF-alpha gene expression in macrophages: regulation by NF-kappa B is independent of c-Jun or C/EBP beta.

The interaction of transcription factors is critical in the regulation of gene expression. This study characterized the mechanism by which NF-kappa B family members interact to regulate the human TNF-alpha gene. A 120-bp TNF-alpha promoter-reporter, possessing binding sites for NF-kappa B (kappa B3), C/EBP beta (CCAAT/enhancer binding protein beta), and c-Jun, was activated by cotransfection of plasmids expressing the wild-type version of each of these transcription factors. Employing adenoviral vectors, dominant-negative versions of NF-kappa B p65, and c-Jun, but not C/EBP beta, suppressed (p < 0.05-0.001) LPS-induced TNF-alpha secretion in primary human macrophages. Following LPS stimulation, NF-kappa B p50/p65 heterodimers bound to the kappa B3 site and c-Jun to the -103 AP-1 site of the TNF-alpha promoter. By transient transfection, NF-kappa B p65 and p50 synergistically activated the TNF-alpha promoter. In contrast, no synergy was observed between NF-kappa B p65, with or without NF-kappa B p50, and c-Jun or C/EBP beta, even in the presence of the coactivator p300. The contribution of the upstream kappa B binding sites was also examined. Following LPS stimulation, the kappa B1 site bound both NF-kappa B p50/p65 heterodimers and p50 homodimers. The binding by NF-kappa B p50 homodimers to the kappa B1, but not to the kappa 3, site contributed to the inability of macrophages to respond to a second LPS challenge. In summary, adjacent kappa B3 and AP-1 sites in the human TNF-alpha promoter contribute independently to LPS-induced activation. Although both the kappa B1 and kappa B3 sites bound transcriptionally active NF-kappa B p50/p65 heterodimers, only the kappa B1 site contributed to down-regulation by NF-kappa B p50 homodimers.

Regulation of NF-kappaB RelA phosphorylation and transcriptional activity by p21(ras) and protein kinase Czeta in primary endothelial cells.

The activity of the transcription factor NF-kappaB is thought to be regulated mainly through cytoplasmic retention by IkappaB molecules. Here we present evidence of a second mechanism of regulation acting on NF-kappaB after release from IkappaB. In endothelial cells this mechanism involves phosphorylation of the RelA subunit of NF-kappaB through a pathway involving activation of protein kinase Czeta (PKCzeta) and p21(ras). We show that transcriptional activity of RelA is dependent on phosphorylation of the N-terminal Rel homology domain but not the C-terminal transactivation domain. Inhibition of phosphorylation by dominant negative mutants of PKCzeta or p21(ras) results in loss of RelA transcriptional activity without interfering with DNA binding. Raf/MEK, small GTPases, phosphatidylinositol 3-kinase, and stress-activated protein kinase pathways are not involved in this mechanism of regulation.

Rejection of cardiac xenografts by CD4+ or CD8+ T cells.

We recently showed that brief complement inhibition induces accommodation of hamster cardiac transplants in nude rats. We have reconstituted nude rats carrying an accommodated xenograft with syngeneic CD4+ or CD8+ T cells to investigate the cellular mechanism of xenograft rejection. We show that CD4+ T cells can initiate xenograft rejection (10 +/- 1.7 days) by promoting production of IgG xenoreactive Abs (XAb). These XAb are able to activate complement as well as to mediate Ab-dependent cell-mediated cytotoxicity. Adoptive transfer of these XAb into naive nude rats provoked hyperacute xenograft rejection (38 +/- 13 min). The rejection was significantly (p < 0.001) delayed by cobra venom factor (CVF; 11 +/- 8 h in four of five cases) but was still more rapid than in control nude rats (3.3 +/- 0.5 days). CVF plus NK cell depletion further prolonged survival (>7 days in four of five cases; p < 0.01 vs CVF only). CD8+ T cell-reconstituted nude rats rejected their grafts later (19.4 +/- 5.8 days) and required a larger number of cells for transfer as compared with CD4+ T cell-reconstituted nude rats. However, second xenografts were rejected more rapidly than first xenografts in CD8+ T cell-reconstituted nude rats (9 +/- 2 days), indicating that the CD8+ T cells had been activated. This study demonstrates that CD4+ and CD8+ T cells can both reject xenografts. The CD4+ cells do so at least in part by generation of helper-dependent XAb that act by both complement-dependent and Ab-dependent cell-mediated cytotoxicity mechanisms; the CD8+ cells do so as helper-independent cytotoxic T cells.

Regulated and endothelial cell-specific expression of Fas ligand: an in vitro model for a strategy aiming at inhibiting xenograft rejection.

BACKGROUND: Immunologically privileged sites have been shown to express Fas ligand (FasL) and may protect themselves by inducing apoptosis of infiltrating inflammatory cells. We asked whether the Fas/FasL interaction could be used to protect a xenograft from rejection. We proposed that endothelial cells that are resistant to Fas-mediated killing could be considered as a vehicle for expression of recombinant FasL. METHODS: Based on the tetracycline-regulated expression system, constructs were designed that allow endothelial cell-specific and regulated expression of FasL by placing the tetracycline-dependent transactivator under control of the murine intercellular adhesion molecule-2 promoter. RESULTS: Primary bovine endothelial cells transfected with FasL efficiently killed Fas-expressing cells in a regulated manner. Not only Fas-positive cell lines but also human peripheral blood lymphocytes underwent apoptosis upon exposure to FasL-transfected endothelial cells. CONCLUSION: This in vitro model may provide tools for the generation of transgenic animals to be used as donors for vascularized xenograft transplantation.

Adenovirus-mediated expression of a dominant negative mutant of p65/RelA inhibits proinflammatory gene expression in endothelial cells without sensitizing to apoptosis.

We hypothesized that blocking the induction of proinflammatory genes associated with endothelial cell (EC) activation, by inhibiting the transcription factor nuclear factor kappaB (NF-kappaB), would prolong survival of vascularized xenografts. Our previous studies have shown that inhibition of NF-kappaB by adenovirus-mediated overexpression of I kappaB alpha suppresses the induction of proinflammatory genes in EC. However, I kappaB alpha sensitizes EC to TNF-alpha-mediated apoptosis, presumably by suppressing the induction of the NF-kappaB-dependent anti-apoptotic genes A20, A1, manganese superoxide dismutase (MnSOD), and cellular inhibitor of apoptosis 2. We report here that adenovirus mediated expression of a dominant negative C-terminal truncation mutant of p65/RelA (p65RHD) inhibits the induction of proinflammatory genes, such as E-selectin, ICAM-1, VCAM-1, IL-8, and inducible nitric oxide synthase, in EC as efficiently as does I kappaB alpha. However, contrary to I kappaB alpha, p65RHD does not sensitize EC to TNF-alpha-mediated apoptosis although both inhibitors suppressed the induction of the anti-apoptotic genes A20, A1, and MnSOD equally well. We present evidence that this difference in sensitization of EC to apoptosis is due to the ability of p65RHD, but not I kappaB alpha, to inhibit the constitutive expression of c-myc, a gene involved in the regulation of TNF-alpha-mediated apoptosis. These data demonstrate that it is possible to block the expression of proinflammatory genes during EC activation by targeting NF-kappaB, without sensitizing EC to apoptosis and establishes the role of c-myc in controlling induction of apoptosis during EC activation. Finally, these data provide the basis for a potential approach to suppress EC activation in vivo in transgenic pigs to be used as donors for xenotransplantation.

Expression of heme oxygenase-1 can determine cardiac xenograft survival.

The rejection of concordant xenografts, such as mouse-to-rat cardiac xenografts, is very similar to the delayed rejection of porcine-to-primate discordant xenografts. In concordant models, this type of rejection is prevented by brief complement inhibition by cobra venom factor (CVF) and sustained T-cell immunosuppression by cyclosporin A (CyA). Mouse hearts that survive indefinitely in rats treated with CVF plus CyA express the anti-inflammatory gene heme oxygenase-1 (HO-1) in their endothelial cells and smooth muscle cells. The anti-inflammatory properties of HO-1 are thought to rely on the ability of this enzyme to degrade heme and generate bilirubin, free iron and carbon monoxide. Bilirubin is a potent anti-oxidant, free iron upregulates the transcription of the cytoprotective gene, ferritin, and carbon monoxide is thought to be essential in regulating vascular relaxation in a manner similar to nitric oxide. We show here that the expression of the HO-1 gene is functionally associated with xenograft survival, and that rapid expression of HO-1 in cardiac xenografts can be essential to ensure long-term xenograft survival.

Regulation of monocyte tissue factor activity by allogeneic and xenogeneic endothelial cells.

The regulation of tissue factor (TF) activity by the cell associated tissue factor pathway inhibitor (TFPI) during monocyte (Mo) and endothelial cell (EC) interactions is not fully understood. This report describes co-ordinate induction of TF antigen (TF-Ag) and membrane-associated TFPI-Ag on human Mo following coculture with human aortic (HAEC) or porcine aortic EC (PAEC) or after stimulation with TNFalpha. We show that both allo- and xenogeneic EC induce human Mo-TF antigen in short-term culture. However, the TF activity of TNFalpha-primed Mo is suppressed when these cells are cocultured with HAEC [by 40.3 +/- 6.3% (p<0.02)] or PAEC [by 50.5 +/- 10.6% (p<0.001)]. Antibody (Ab) blocking studies confirm that TFPI is the principal anticoagulant associated with this suppression of TF-activity. Our data indicate that anti-TF activity originates, at least in part, from the activated human Mo in the coculture; additionally, specific generation of TFPI by Mo is observed under the xenogeneic culture conditions. As Mo associated TF, induced by allo- or xenogeneic EC interactions, is regulated by cell-associated TFPI, we propose that infiltrating Mo may modulate the thrombotic process at sites of vascular injury in association with both allo- and xenograft rejection.

Activation of human platelets by the membrane-expressed A1 domain of von Willebrand factor.

Platelet activation and microthrombus formation are invariable features of xenograft rejection and the vascular injury observed when porcine organs are transplanted into primates. This pathological process could be mediated, at least in part, by aberrant interactions of von Willebrand Factor (vWF) associated with the donor vasculature with host platelets. Unlike human vWF, native porcine vWF (pvWF) interacts with human GPIb independently of shear stress or nonphysiological stimuli, eg, ristocetin. We therefore contrasted the potential of isolated human and porcine vWF-A1-domains to interact with human platelets in vitro. Both human and porcine vWF-A1-domains expressed as glycosyl phosphatidylinositol-linked FLAG fusion proteins on COS-7 cells induced GPIb-dependent aggregation and intracellular Ca++ uptake of platelets, independent of both the remainder of the vWF protein and additional modifying factors. Porcine A1-domains were more potent than human homologues, and in addition ristocetin could boost platelet aggregation only with the human A1-domain. Putative conformational changes in the porcine A1-domain could result in the heightened, ristocetin-independent interactions observed with human platelets and may be of importance for xenograft survival.

Glucocorticoids inhibit E-selectin expression by targeting NF-kappaB and not ATF/c-Jun.

E-selectin, an adhesion molecule expressed on the surface of activated endothelial cells, is essential for leukocyte rolling on endothelium which leads to extravasation in the process of inflammation. Induction of E-selectin expression by proinflammatory stimuli such as TNF-alpha or LPS is reduced markedly in the presence of dexamethasone, a synthetic glucocorticoid and potent anti-inflammatory agent. We have investigated the molecular mechanism underlying dexamethasone-mediated E-selectin repression in porcine aortic endothelial cells. Reduced E-selectin protein expression is paralleled by a decrease in E-selectin mRNA and is based on changes in transcription rate. Analysis of the E-selectin promoter revealed that induction by proinflammatory stimuli as well as repression by dexamethasone are mediated by the same promoter region containing three closely spaced binding sites for nuclear factor (NF)-kappaB and an element, NF-ELAM-1 (endothelial leukocyte adhesion molecule-1), constitutively occupied by ATF and c-Jun. NF-ELAM-1 contributes to maximal promoter activity, but does not confer glucocorticoid inhibition, as demonstrated by site-directed mutagenesis. In contrast, transcription directed by the E-selectin NF-kappaB elements is reduced strongly in the presence of dexamethasone, thus identifying NF-kappaB as the primary target for glucocorticoid-mediated E-selectin repression.

Effect of porcine endothelial tissue factor pathway inhibitor on human coagulation factors.

BACKGROUND: Delayed xenograft rejection (DXR) is characterized by inflammation and vascular thrombosis. Activation of coagulation may occur as a result of tissue factor (TF) expression on both activated donor endothelial cells (EC) and recipient infiltrating monocytes (Mo). In addition, natural anticoagulants associated with porcine endothelial cells may not function adequately across species. METHODS: In the present study, we examined the interaction of the TF pathway of coagulation with the natural anticoagulant TF pathway inhibitor, in xenogeneic leukocyte-EC cultures in vitro, and during rejection of discordant xenografts in vivo. RESULTS: Coculture of human Mo with pig aortic EC (PAEC) resulted in 1.7-fold and 2-fold higher induction of Mo TF and Mo intercellular adhesion molecule-1, respectively, when compared with coculture with human aortic endothelial cells (HAEC). In addition, TF-dependent and -independent activation of coagulation factor X was higher on PAEC than on HAEC. Low levels of mRNA for tissue factor pathway inhibitor (TFPI) and its variant, TFPI-2, in resting PAEC were up-regulated by stimulation with tumor necrosis factor alpha. Procoagulant activity of recombinant human TF complexed to activated factor VII was inhibited by PAEC and HAEC-associated TFPI by 22% and 56%, respectively. In contrast, human activated factor X (factor Xa) activity was inhibited by human, but not porcine, EC-associated TFPI, suggesting functional incompatibility of PAEC for human factor Xa. Endothelial TFPI was detected in pig control organs and after hyperacute rejection, but was lost from the vasculature during DXR. CONCLUSIONS: Lack of appropriate human factor Xa inhibition by porcine EC during hyperacute rejection and loss of porcine EC TFPI during DXR could promote the development of a procoagulant environment leading to xenograft rejection.

Inhibition of bovine endothelial cell activation in vitro by regulated expression of a transdominant inhibitor of NF-kappa B.

The activation of endothelial cells is a recurrent phenomenon linked to pathologic conditions such as inflammation, chronic arthritis, allo- and xenograft rejection. To inhibit endothelial cell activation we have constructed a transactivation-deficient derivative of the p65/RelA subunit of NF-kappa B, a transcription factor known to be crucial for the induction of adhesion molecules, cytokines and procoagulants in activated endothelial cells. This protein (p65RHD) comprises the Rel homology domain of the RelA subunit, retaining dimerization, DNA binding, and nuclear localization functions, but is deficient in transcriptional activation, and acts as a competitive inhibitor of NF-kappa B. Our data demonstrate that p65RHD is a potent and specific inhibitor of NF-kappa B-mediated induction of a number of genes, such as I kappa B alpha, IL-8, E-selectin, P-selectin, and tissue factor in endothelial cells. Furthermore, tetracycline-inducible expression of p65RHD in stably transfected primary endothelial cells inhibits the induction of gene expression equally well. This regulated system of gene expression provides the basis for a novel therapeutic approach to the pathologic effects of endothelial cell activation, especially in delayed xenograft rejection, by using transgenic animals as organ donors.

Identification and characterization of CD39/vascular ATP diphosphohydrolase.

Vascular ATP diphosphohydrolase (ATPDase) is a plasma membrane-bound enzyme that hydrolyses extracellular ATP and ADP to AMP. Analysis of amino acid sequences available from various mammalian and avian ATPDases revealed their close homology with CD39, a putative B-cell activation marker. We, therefore, isolated CD39 cDNA from human endothelial cells and expressed this in COS-7 cells. CD39 was found to have both immunological identity to, and functional characteristics of, the vascular ATPDase. We also demonstrated that ATPDase could inhibit platelet aggregation in response to ADP, collagen, and thrombin, and that this activity in transfected COS-7 cells was lost following exposure to oxidative stress. ATPDase mRNA was present in human placenta, lung, skeletal muscle, kidney, and heart and was not detected in brain. Multiple RNA bands were detected with the CD39 cDNA probe that most probably represent different splicing products. Finally, we identified an unique conserved motif, DLGGASTQ, that could be crucial for nucleotide binding, activity, and/or structure of ATPDase. Because ATPDase activity is lost with endothelial cell activation, overexpression of the functional enzyme, or a truncated mutant thereof, may prevent platelet activation associated with vascular inflammation.

The intron-exon structure of the porcine E-selectin-encoding gene.

We have cloned and sequenced the gene encoding porcine E-selectin. The gene comprises 12 exons and 11 introns. Two pseudoexons are contained within intron 4 and intron 6. These sequences are similar to the corresponding exons in the human E-selectin sequence; however, they are not present in the porcine E-selectin-encoding cDNA. Transcription starts at position -498 relative to the translation initiation site. The first ATG is located within exon 2. Translation stops in exon 11 leaving exon 12 untranslated in its entirety.

Glucocorticoid-mediated repression of NFkappaB activity in endothelial cells does not involve induction of IkappaBalpha synthesis.

Repression of NFkappaB-dependent gene expression is one of the major elements of immunosuppression by glucocorticoids. Protein-protein interactions between the glucocorticoid receptor and NFkappaB have been characterized and shown to be a possible mechanism of mutual inhibition of transactivation properties. More recently, glucocorticoid-mediated induction of IkappaBalpha, an inhibitor of NFkappaB, has been described in monocytes and lymphocytes; an increase in IkappaBalpha mRNA and protein resulted in inactivation and cytosolic retention of NFkappaB. Thus, rather than the physical interaction between the glucocorticoid receptor and NFkappaB, the up-regulation of IkappaBalpha was presented as the key element in immunosuppression by glucocorticoids. In contrast, we show that the IkappaBalpha pathway is not involved in glucocorticoid-mediated inhibition of NFkappaB activity in endothelial cells. Although transcriptional activation by NFkappaB was significantly reduced in the presence of glucocorticoids, we did not detect induction of IkappaBalpha protein that could prevent nuclear translocation of NFkappaB upon stimulation with lipopolysaccharide or tumor necrosis factor alpha. Furthermore, treatment with glucocorticoids did not seem to affect the transcription rate or mRNA stability of IkappaBalpha. We therefore conclude that, although induction of IkappaBalpha expression by glucocorticoids seems to be of importance in monocytes and lymphocytes, it cannot explain inhibition of NFkappaB-dependent gene expression in endothelial cells. Our results emphasize the relevance of physical interaction between the glucocorticoid receptor and NFkappaB in endothelial cells and thus in suppression of inflammation by glucocorticoids.