Uncoupling Caveolae From Intracellular Signaling In Vivo.

RATIONALE: Caveolin-1 (Cav-1) negatively regulates endothelial nitric oxide (NO) synthase-derived NO production, and this has been mapped to several residues on Cav-1, including F92. Herein, we reasoned that endothelial expression of an F92ACav-1 transgene would let us decipher the mechanisms and relationships between caveolae structure and intracellular signaling. OBJECTIVE: This study was designed to separate caveolae formation from its downstream signaling effects. METHODS AND RESULTS: An endothelial-specific doxycycline-regulated mouse model for the expression of Cav-1-F92A was developed. Blood pressure by telemetry and nitric oxide bioavailability by electron paramagnetic resonance and phosphorylation of vasodilator-stimulated phosphoprotein were determined. Caveolae integrity in the presence of Cav-1-F92A was measured by stabilization of caveolin-2, sucrose gradient, and electron microscopy. Histological analysis of heart and lung, echocardiography, and signaling were performed. CONCLUSIONS: This study shows that mutant Cav-1-F92A forms caveolae structures similar to WT but leads to increases in NO bioavailability in vivo, thereby demonstrating that caveolae formation and downstream signaling events occur through independent mechanisms.

Reperfusion injury intensifies the adaptive human T cell alloresponse in a human-mouse chimeric artery model.

OBJECTIVE: Perioperative nonimmune injuries to an allograft can decrease graft survival. We have developed a model for studying this process using human materials. METHODS AND RESULTS: Human artery segments were transplanted as infrarenal aortic interposition grafts into an immunodeficient mouse host, allowed to "heal in" for 30 days, and then retransplanted into a second mouse host. To induce a reperfusion injury, the healed-in artery segments were incubated for 3 hours under hypoxic conditions ex vivo before retransplantation. To induce immunologic rejection, the animals receiving the retransplanted artery segment were adoptively transferred with human peripheral blood mononuclear cells or purified T cells from a donor allogeneic to the artery 1 week before surgery. To compare rejection of injured versus healthy tissues, these manipulations were combined. Results were analyzed ex vivo by histology, morphometry, immunohistochemistry, and mRNA quantitation or in vivo by ultrasound. Our results showed that reperfusion injury, which otherwise heals with minimal sequelae, intensifies the degree of allogeneic T cell-mediated injury to human artery segments. CONCLUSIONS: We developed a new human-mouse chimeric model demonstrating interactions of reperfusion injury and alloimmunity using human cells and tissues that may be adapted to study other forms of nonimmune injury and other types of adaptive immune responses.

Cellularity and structure of fresh human coronary thrombectomy specimens; presence of cells with markers of progenitor cells.

Acute coronary syndromes and acute myocardial infarctions are often related to plaque rupture and the formation of thrombi at the site of the rupture. We examined fresh coronary thrombectomy specimens from patients with acute coronary syndromes and assessed their structure and cellularity. The thrombectomy specimens consisted of platelets, erythrocytes and inflammatory cells. Several specimens contained multiple cholesterol crystals. Culture of thrombectomy specimens yielded cells growing in various patterns depending on the culture medium used. Culture in serum-free stem cell enrichment medium yielded cells with features of endothelial progenitor cells which survived in culture for a year. Immunohistochemical analysis of the thrombi revealed cells positive for CD34, cells positive for CD15 and cells positive for desmin in situ, whereas cultured cell from thrombi was desmin positive but pancytokeratin negative. Cells cultured in endothelial cell medium were von Willebrand factor positive. The content of coronary thrombectomy specimens is heterogeneous and consists of blood cells but also possibly cells from the vascular wall and cholesterol crystals. The culture of cells contained in the specimens yielded multiplying cells, some of which demonstrated features of haematopoietic progenitor cells and which differentiated into various cell-types.

Reticulon 4B (Nogo-B) is a novel regulator of hepatic fibrosis.

UNLABELLED: Nogo-B, also known as Reticulon 4B, plays important roles in vascular injuries. Its function in the liver is not understood. The aim of this study was to characterize Nogo-B in liver fibrosis and cirrhosis. Nogo-B distribution was assessed in normal and cirrhotic human liver sections. We also determined the levels of liver fibrosis in wild-type (WT) and Nogo-A/B knockout (NGB KO) mice after sham operation or bile duct ligation (BDL). To investigate the mechanisms of Nogo-B's involvement in fibrosis, hepatic stellate cells were isolated from WT and NGB KO mice and transformed into myofibroblasts. Portal pressure was measured to test whether Nogo-B gene deletion could ameliorate portal hypertension. In normal livers, Nogo-B expression was found in nonparenchymal cells, whereas its expression in hepatocytes was minimal. Nogo-B staining was significantly elevated in cirrhotic livers. Fibrosis was significantly increased in WT mice 4 weeks after BDL compared with NGB KO mice. The absence of Nogo-B significantly reduced phosphorylation of Smad2 levels upon transforming growth factor ß (TGF-ß) stimulation. Reconstitution of the Nogo-B gene into NGB KO fibroblasts restored Smad2 phosphorylation. Four weeks after BDL, portal pressure was significantly increased in WT mice by 47%, compared with sham-operated controls (P = 0.03), whereas such an increase in portal pressure was not observed in NGB KO mice (P = NS). CONCLUSION: Nogo-B regulates liver fibrosis, at least in part, by facilitating the TGFß/Smad2 signaling pathway in myofibroblasts. Because absence of Nogo-B ameliorates liver fibrosis and portal hypertension, Nogo-B blockade may be a potential therapeutic target in fibrosis/cirrhosis.

Reticulon 4B (Nogo-B) is necessary for macrophage infiltration and tissue repair.

Blood vessel formation during ischemia and wound healing requires coordination of the inflammatory response with genes that regulate blood vessel assembly. Here we show that the reticulon family member 4B, aka Nogo-B, is upregulated in response to ischemia and is necessary for blood flow recovery secondary to ischemia and wound healing. Mice lacking Nogo-B exhibit reduced arteriogenesis and angiogenesis that are linked to a decrease in macrophage infiltration and inflammatory gene expression in vivo. Bone marrow-derived macrophages isolated from Nogo knock-out mice have reduced spreading and chemotaxis due to impaired Rac activation. Bone marrow reconstitution experiments show that Nogo in myeloid cells is necessary to promote macrophage homing and functional recovery after limb ischemia. Thus, endogenous Nogo coordinates macrophage-mediated inflammation with arteriogenesis, wound healing, and blood flow control.

Urocortin2 inhibits tumor growth via effects on vascularization and cell proliferation.

The corticotropin-releasing factor (CRF) receptor CRFR2 is expressed widely in peripheral tissues and in the vasculature, although its functional roles in those tissues have only recently begun to be elucidated. Previously we found that genetic deletion of CRFR2 resulted in profound postnatal hypervascularization in mice, characterized by both an increase in total vessel number and a dramatic increase in vessel diameter. These data strongly suggested that ligands for CRFR2 act to limit tissue vascularity, perhaps as a counterbalance to factors that promote neovascularization. Urocortin 2 (Ucn2) is a specific ligand for the CRFR2. We hypothesized that activation of CRFR2 by Ucn2 might thus suppress tumor vascularization and consequently limit tumor growth. Here, we show that viral-mediated expression of Ucn2 strikingly inhibits the growth and vascularization of Lewis Lung Carcinoma Cell (LLCC) tumors in vivo. Further, we found that this effect on tumor growth inhibition was independent of whether exposure to Ucn2 occurred before or after establishment of measurable tumors. In vitro, Ucn2 directly inhibited the proliferation of LLCC, suggesting that the tumor-suppressing effects of CRFR2 activation involve a dual mechanism of both a direct inhibition of tumor cell cycling and the suppression of tumor vascularization. These results establish that Ucn2 inhibits tumor growth, suggesting a potential therapeutic role for CRFR2 ligands in clinical malignancies.

An insight into the sialome of Glossina morsitans morsitans.

BACKGROUND: Blood feeding evolved independently in worms, arthropods and mammals. Among the adaptations to this peculiar diet, these animals developed an armament of salivary molecules that disarm their host's anti-bleeding defenses (hemostasis), inflammatory and immune reactions. Recent sialotranscriptome analyses (from the Greek sialo = saliva) of blood feeding insects and ticks have revealed that the saliva contains hundreds of polypeptides, many unique to their genus or family. Adult tsetse flies feed exclusively on vertebrate blood and are important vectors of human and animal diseases. Thus far, only limited information exists regarding the Glossina sialome, or any other fly belonging to the Hippoboscidae. RESULTS: As part of the effort to sequence the genome of Glossina morsitans morsitans, several organ specific, high quality normalized cDNA libraries have been constructed, from which over 20,000 ESTs from an adult salivary gland library were sequenced. These ESTs have been assembled using previously described ESTs from the fat body and midgut libraries of the same fly, thus totaling 62,251 ESTs, which have been assembled into 16,743 clusters (8,506 of which had one or more EST from the salivary gland library). Coding sequences were obtained for 2,509 novel proteins, 1,792 of which had at least one EST expressed in the salivary glands. Despite library normalization, 59 transcripts were overrepresented in the salivary library indicating high levels of expression. This work presents a detailed analysis of the salivary protein families identified. Protein expression was confirmed by 2D gel electrophoresis, enzymatic digestion and mass spectrometry. Concurrently, an initial attempt to determine the immunogenic properties of selected salivary proteins was undertaken. CONCLUSIONS: The sialome of G. m. morsitans contains over 250 proteins that are possibly associated with blood feeding. This set includes alleles of previously described gene products, reveals new evidence that several salivary proteins are multigenic and identifies at least seven new polypeptide families unique to Glossina. Most of these proteins have no known function and thus, provide a discovery platform for the identification of novel pharmacologically active compounds, innovative vector-based vaccine targets, and immunological markers of vector exposure.

Proventriculus-specific cDNAs characterized from the tsetse, Glossina morsitans morsitans.

Peritrophic matrix (peritrophic membrane or PM) is an important structure in the gut of most insects at some stage in their development. It is composed of chitin, proteins and proteoglycans. Multiple roles for the PM ranging from partitioning of digestive enzymes and food to protection of gut epithelial cells from viral and parasitic invasion have been proposed. While most adult members of Diptera have a Type I PM synthesized in response to a blood meal, the medically and agriculturally important vector insect, tsetse has a sleeve-like Type II PM which is constitutively synthesized by cells in the proventriculus (cardia). Using a differential hybridization approach, we have identified three abundant cDNAs from a proventriculus cDNA library of GLOSSINA MORSITANS MORSITANS: GmPro1, GmPro2 and GmPro3. DNA sequence analysis indicates that GmPro1 and GmPro2 share similarities with the peritrophin-15 family of larval PM proteins, while GmPro3 is a member of the serine protease family. Northern analysis indicates that transcripts for all three cDNAs are preferentially expressed in the proventriculus tissue. The expression profile of these genes in response to the presence of trypanosome indicates that transcription of GmPro1 is increased in the presence of parasites (immune sensitive), while the other two are not affected. Western analysis using antibodies developed against the recombinant GmPro2 shows its primary localization in the gut to be within the peritrophic matrix structure. We discuss the molecular characteristics of these proventriculus specific cDNAs and their products as well as their potential role for vector control studies.

Proventriculus (cardia) plays a crucial role in immunity in tsetse fly (Diptera: Glossinidiae).

Fat body and hemocytes play a central role in cellular and humoral responses for systemic infections in invertebrates, similar to the mammalian liver and blood cells. Epithelial surfaces, in particular the midgut, participate in the initial local immune responses in order to aid in the generation of the terminal cytotoxic molecules that mediate non-self recognition. Here, we describe for the first time the immune responses of a cluster of cells at the foregut/midgut junction--known as proventriculus (cardia) in the medically and agriculturally important insect, tsetse fly (Diptera: Glossinidae). We provide evidence for the transcriptional induction of the antimicrobial peptides attacin and defensin as well as for the reactive nitrogen intermediate (RNI) nitric oxide synthase (NOS) upon microbial challenge by either microinjection or feeding. Proventriculus from immune challenged flies also has higher NOS and nitric oxide (NO) activities as well as increased levels of the reactive oxygen intermediate (ROI), hydrogen peroxide (H2O2). In several vector pathogen systems, including tsetse flies and African trypanosomes, stimulation of systemic responses prior to pathogen acquisition has been shown to reduce disease transmission. Furthermore, the induction of systemic immune responses has been documented while pathogens are still differentiating within the midgut environment. While evidence for a close molecular communication between the local and systemic responses is accumulating, the molecular signals that mediate these interactions are at present unknown. Reactive intermediates such as NO or H2O2 may function as immunological signals for mediating the molecular communication between the different insect compartments. We discuss the putative role of the proventriculus in invertebrate immunity and specifically speculate on its significance for trypanosome transmission in tsetse.

What can we hope to gain for trypanosomiasis control from molecular studies on tsetse biology ?

At times of crisis when epidemics rage and begin to take their toll on affected populations, as we have been witnessing with African trypanosomiasis in subSahara, the dichotomy of basic versus applied research deepens. While undoubtedly the treatment of thousands of infected people is the top priority, without continued research and development on the biology of disease agents and on ecological and evolutionary forces impacting these epidemics, little progress can be gained in the long run for the eventual control of these diseases. Here, we argue the need for additional research in one under-investigated area, that is the biology of the tsetse vector. Lacking are studies aimed to understand the genetic and cellular basis of tsetse interactions with trypanosomes as well as the genetic and biochemical basis of its ability to transmit these parasites. We discuss how this knowledge has the potential to contribute to the development of new vector control strategies as well as to improve the efficacy and affordability of the existing control approaches.

The role of Nogo and the mitochondria-endoplasmic reticulum unit in pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is caused by excessive proliferation of vascular cells, which occlude the lumen of pulmonary arteries (PAs) and lead to right ventricular failure. The cause of the vascular remodeling in PAH remains unknown, and the prognosis of PAH remains poor. Abnormal mitochondria in PAH PA smooth muscle cells (SMCs) suppress mitochondria-dependent apoptosis and contribute to the vascular remodeling. We hypothesized that early endoplasmic reticulum (ER) stress, which is associated with clinical triggers of PAH including hypoxia, bone morphogenetic protein receptor II mutations, and HIV/herpes simplex virus infections, explains the mitochondrial abnormalities and has a causal role in PAH. We showed in SMCs from mice that Nogo-B, a regulator of ER structure, was induced by hypoxia in SMCs of the PAs but not the systemic vasculature through activation of the ER stress-sensitive transcription factor ATF6. Nogo-B induction increased the distance between the ER and mitochondria and decreased ER-to-mitochondria phospholipid transfer and intramitochondrial calcium. In addition, we noted inhibition of calcium-sensitive mitochondrial enzymes, increased mitochondrial membrane potential, decreased mitochondrial reactive oxygen species, and decreased mitochondria-dependent apoptosis. Lack of Nogo-B in PASMCs from Nogo-A/B-/- mice prevented these hypoxia-induced changes in vitro and in vivo, resulting in complete resistance to PAH. Nogo-B in the serum and PAs of PAH patients was also increased. Therefore, triggers of PAH may induce Nogo-B, which disrupts the ER-mitochondria unit and suppresses apoptosis. This could rescue PASMCs from death during ER stress but enable the development of PAH through overproliferation. The disruption of the ER-mitochondria unit may be relevant to other diseases in which Nogo is implicated, such as cancer or neurodegeneration.