Molecular basis of atypicality of bupropion inferred from its receptor engagement in nervous system tissues.

Despite decades of clinical use and research, the mechanism of action (MOA) of antidepressant medications remains poorly understood. Selective serotonin reuptake inhibitors (SSRIs) and serotonin-norepinephrine reuptake inhibitors (SNRIs) are the most commonly prescribed antidepressants-atypical antidepressants such as bupropion have also proven effective, while exhibiting a divergent clinical phenotype. The difference in phenotypic profiles presumably lies in the differences among the MOAs of SSRIs/SNRIs and bupropion. We integrated the ensemble of bupropion's affinities for all its receptors with the expression levels of those targets in nervous system tissues. This "combined target tissue" profile of bupropion was compared to those of duloxetine, fluoxetine, and venlafaxine to isolate the unique target tissue effects of bupropion. Our results suggest that the three monoamines-serotonin, norepinephrine, and dopamine-all contribute to the common antidepressant effects of SSRIs, SNRIs, and bupropion. At the same time, bupropion is unique in its action on 5-HT3AR in the dorsal root ganglion and nicotinic acetylcholine receptors in the pineal gland. These unique tissue-specific activities may explain unique therapeutic effects of bupropion, such as pain management and smoking cessation, and, given melatonin's association with nicotinic acetylcholine receptors and depression, highlight the underappreciated role of the melatonergic system in bupropion's MOA.

Rare Synaptogenesis-Impairing Mutations in SLITRK5 Are Associated with Obsessive Compulsive Disorder.

Obsessive compulsive disorder (OCD) is substantially heritable, but few molecular genetic risk factors have been identified. Knockout mice lacking SLIT and NTRK-Like Family, Member 5 (SLITRK5) display OCD-like phenotypes including serotonin reuptake inhibitor-sensitive pathologic grooming, and corticostriatal dysfunction. Thus, mutations that impair SLITRK5 function may contribute to the genetic risk for OCD. We re-sequenced the protein-coding sequence of the human SLITRK5 gene (SLITRK5) in three hundred and seventy seven OCD subjects and compared rare non-synonymous mutations (RNMs) in that sample with similar mutations in the 1000 Genomes database. We also performed in silico assessments and in vitro functional synaptogenesis assays on the Slitrk5 mutations identified. We identified four RNM's among these OCD subjects. There were no significant differences in the prevalence or in silico effects of rare non-synonymous mutations in the OCD sample versus controls. Direct functional testing of recombinant SLITRK5 proteins found that all mutations identified in OCD subjects impaired synaptogenic activity whereas none of the pseudo-matched mutations identified in 1000 Genomes controls had significant effects on SLITRK5 function (Fisher's exact test P = 0.028). These results demonstrate that rare functional mutations in SLITRK5 contribute to the genetic risk for OCD in human populations. They also highlight the importance of biological characterization of allelic effects in understanding genotype-phenotype relationships as there were no statistical differences in overall prevalence or bioinformatically predicted effects of OCD case versus control mutations. Finally, these results converge with others to highlight the role of aberrant synaptic function in corticostriatal neurons in the pathophysiology of OCD.

Slitrk5 Mediates BDNF-Dependent TrkB Receptor Trafficking and Signaling.

Recent studies in humans and in genetic mouse models have identified Slit- and NTRK-like family (Slitrks) as candidate genes for neuropsychiatric disorders. All Slitrk isotypes are highly expressed in the CNS, where they mediate neurite outgrowth, synaptogenesis, and neuronal survival. However, the molecular mechanisms underlying these functions are not known. Here, we report that Slitrk5 modulates brain-derived neurotrophic factor (BDNF)-dependent biological responses through direct interaction with TrkB receptors. Under basal conditions, Slitrk5 interacts primarily with a transsynaptic binding partner, protein tyrosine phosphatase δ (PTPδ); however, upon BDNF stimulation, Slitrk5 shifts to cis-interactions with TrkB. In the absence of Slitrk5, TrkB has a reduced rate of ligand-dependent recycling and altered responsiveness to BDNF treatment. Structured illumination microscopy revealed that Slitrk5 mediates optimal targeting of TrkB receptors to Rab11-positive recycling endosomes through recruitment of a Rab11 effector protein, Rab11-FIP3. Thus, Slitrk5 acts as a TrkB co-receptor that mediates its BDNF-dependent trafficking and signaling.

Historeceptomic Fingerprints for Drug-Like Compounds.

Most drugs exert their beneficial and adverse effects through their combined action on several different molecular targets (polypharmacology). The true molecular fingerprint of the direct action of a drug has two components: the ensemble of all the receptors upon which a drug acts and their level of expression in organs/tissues. Conversely, the fingerprint of the adverse effects of a drug may derive from its action in bystander tissues. The ensemble of targets is almost always only partially known. Here we describe an approach improving upon and integrating both components: in silico identification of a more comprehensive ensemble of targets for any drug weighted by the expression of those receptors in relevant tissues. Our system combines more than 300,000 experimentally determined bioactivity values from the ChEMBL database and 4.2 billion molecular docking scores. We integrated these scores with gene expression data for human receptors across a panel of human tissues to produce drug-specific tissue-receptor (historeceptomics) scores. A statistical model was designed to identify significant scores, which define an improved fingerprint representing the unique activity of any drug. These multi-dimensional historeceptomic fingerprints describe, in a novel, intuitive, and easy to interpret style, the holistic, in vivo picture of the mechanism of any drug's action. Valuable applications in drug discovery and personalized medicine, including the identification of molecular signatures for drugs with polypharmacologic modes of action, detection of tissue-specific adverse effects of drugs, matching molecular signatures of a disease to drugs, target identification for bioactive compounds with unknown receptors, and hypothesis generation for drug/compound phenotypes may be enabled by this approach. The system has been deployed at drugable.org for access through a user-friendly web site.

Slitrks as emerging candidate genes involved in neuropsychiatric disorders.

Slitrks are a family of structurally related transmembrane proteins belonging to the leucine-rich repeat (LRR) superfamily. Six family members exist (Slitrk1-6) and all are highly expressed in the central nervous system (CNS). Slitrks have been implicated in mediating basic neuronal processes, ranging from neurite outgrowth and dendritic elaboration to neuronal survival. Recent studies in humans and genetic mouse models have led to the identification of Slitrks as candidate genes that might be involved in the development of neuropsychiatric conditions, such as obsessive compulsive spectrum disorders and schizophrenia. Although these system-level approaches have suggested that Slitrks play prominent roles in CNS development, key questions remain regarding the molecular mechanisms through which they mediate neuronal signaling and connectivity.

Slitrk5 deficiency impairs corticostriatal circuitry and leads to obsessive-compulsive-like behaviors in mice.

Obsessive-compulsive disorder (OCD) is a common psychiatric disorder defined by the presence of obsessive thoughts and repetitive compulsive actions, and it often encompasses anxiety and depressive symptoms. Recently, the corticostriatal circuitry has been implicated in the pathogenesis of OCD. However, the etiology, pathophysiology and molecular basis of OCD remain unknown. Several studies indicate that the pathogenesis of OCD has a genetic component. Here we demonstrate that loss of a neuron-specific transmembrane protein, SLIT and NTRK-like protein-5 (Slitrk5), leads to OCD-like behaviors in mice, which manifests as excessive self-grooming and increased anxiety-like behaviors, and is alleviated by the selective serotonin reuptake inhibitor fluoxetine. Slitrk5(-/-) mice show selective overactivation of the orbitofrontal cortex, abnormalities in striatal anatomy and cell morphology and alterations in glutamate receptor composition, which contribute to deficient corticostriatal neurotransmission. Thus, our studies identify Slitrk5 as an essential molecule at corticostriatal synapses and provide a new mouse model of OCD-like behaviors.

Angiomodulin is a specific marker of vasculature and regulates vascular endothelial growth factor-A-dependent neoangiogenesis.

Blood vessel formation is controlled by the balance between pro- and antiangiogenic pathways. Although much is known about the factors that drive sprouting of neovessels, the factors that stabilize and pattern neovessels are undefined. The expression of angiomodulin (AGM), a vascular endothelial growth factor (VEGF)-A binding protein, was increased in the vasculature of several human tumors as compared to normal tissue, raising the hypothesis that AGM may modulate VEGF-A-dependent vascular patterning. To elucidate the expression pattern of AGM, we developed an AGM knockin reporter mouse (AGM(lacZ/+)), with which we demonstrate that AGM is predominantly expressed in the vasculature of developing embryos and adult organs. During physiological and pathological angiogenesis, AGM is upregulated in the angiogenic vasculature. Using the zebrafish model, we found that AGM is restricted to developing vasculature by 17 to 22 hours postfertilization. Blockade of AGM activity with morpholino oligomers results in prominent angiogenesis defects in vascular sprouting and remodeling. Concurrent knockdown of both AGM and VEGF-A results in synergistic angiogenesis defects. When VEGF-A is overexpressed, the compensatory induction of the VEGF-A receptor, VEGFR2/flk-1, is blocked by the simultaneous injection of AGM morpholino oligomers. These results demonstrate that the vascular-specific marker AGM modulates vascular remodeling in part by temporizing the proangiogenic effects of VEGF-A.

Generation of a functional and durable vascular niche by the adenoviral E4ORF1 gene.

Vascular cells contribute to organogenesis and tumorigenesis by producing unknown factors. Primary endothelial cells (PECs) provide an instructive platform for identifying factors that support stem cell and tumor homeostasis. However, long-term maintenance of PECs requires stimulation with cytokines and serum, resulting in loss of their angiogenic properties. To circumvent this hurdle, we have discovered that the adenoviral E4ORF1 gene product maintains long-term survival and facilitates organ-specific purification of PECs, while preserving their vascular repertoire for months, in serum/cytokine-free cultures. Lentiviral introduction of E4ORF1 into human PECs (E4ORF1(+) ECs) increased the long-term survival of these cells in serum/cytokine-free conditions, while preserving their in vivo angiogenic potential for tubulogenesis and sprouting. Although E4ORF1, in the absence of mitogenic signals, does not induce proliferation of ECs, stimulation with VEGF-A and/or FGF-2 induced expansion of E4ORF1(+) ECs in a contact-inhibited manner. Indeed, VEGF-A-induced phospho MAPK activation of E4ORF1(+) ECs is comparable with that of naive PECs, suggesting that the VEGF receptors remain functional upon E4ORF1 introduction. E4ORF1(+) ECs inoculated in implanted Matrigel plugs formed functional, patent, humanized microvessels that connected to the murine circulation. E4ORF1(+) ECs also incorporated into neo-vessels of human tumor xenotransplants and supported serum/cytokine-free expansion of leukemic and embryonal carcinoma cells. E4ORF1 augments survival of PECs in part by maintaining FGF-2/FGF-R1 signaling and through tonic Ser-473 phosphorylation of Akt, thereby activating the mTOR and NF-kappaB pathways. Therefore, E4ORF1(+) ECs establish an Akt-dependent durable vascular niche not only for expanding stem and tumor cells but also for interrogating the roles of vascular cells in regulating organ-specific vascularization and tumor neo-angiogenesis.

CD133 expression is not restricted to stem cells, and both CD133+ and CD133- metastatic colon cancer cells initiate tumors.

Colon cancer stem cells are believed to originate from a rare population of putative CD133+ intestinal stem cells. Recent publications suggest that a small subset of colon cancer cells expresses CD133, and that only these CD133+ cancer cells are capable of tumor initiation. However, the precise contribution of CD133+ tumor-initiating cells in mediating colon cancer metastasis remains unknown. Therefore, to temporally and spatially track the expression of CD133 in adult mice and during tumorigenesis, we generated a knockin lacZ reporter mouse (CD133lacZ/+), in which the expression of lacZ is driven by the endogenous CD133 promoters. Using this model and immunostaining, we discovered that CD133 expression in colon is not restricted to stem cells; on the contrary, CD133 is ubiquitously expressed on differentiated colonic epithelium in both adult mice and humans. Using Il10-/-CD133lacZ mice, in which chronic inflammation in colon leads to adenocarcinomas, we demonstrated that CD133 is expressed on a full gamut of colonic tumor cells, which express epithelial cell adhesion molecule (EpCAM). Similarly, CD133 is widely expressed by human primary colon cancer epithelial cells, whereas the CD133- population is composed mostly of stromal and inflammatory cells. Conversely, CD133 expression does not identify the entire population of epithelial and tumor-initiating cells in human metastatic colon cancer. Indeed, both CD133+ and CD133- metastatic tumor subpopulations formed colonospheres in in vitro cultures and were capable of long-term tumorigenesis in a NOD/SCID serial xenotransplantation model. Moreover, metastatic CD133- cells form more aggressive tumors and express typical phenotypic markers of cancer-initiating cells, including CD44 (CD44+CD24-), whereas the CD133+ fraction is composed of CD44lowCD24+ cells. Collectively, our data suggest that CD133 expression is not restricted to intestinal stem or cancer-initiating cells, and during the metastatic transition, CD133+ tumor cells might give rise to the more aggressive CD133(- )subset, which is also capable of tumor initiation in NOD/SCID mice.

Niche players: spermatogonial progenitors marked by GPR125.

The undifferentiated spermatogonia of adult mouse testes are composed of both true stem cells and committed progenitors. It is unclear what normally prevents these adult germ cells from manifesting multipotency. The critical elements of the spermatogonial stem cell niche, while poorly understood, are thought to be composed of Sertoli cells with several other somatic cell types in close proximity. We recently discovered a novel orphan G-protein coupled receptor (GPR125) that is restricted to undifferentiated spermatogonia within the testis. GPR125 expression was maintained when the progenitor cells were extracted from the in vivo niche and propagated under growth conditions that recapitulate key elements of the niche. Such conditions preserved the ability of the cells to generate multipotent derivatives, known as multipotent adult spermatogonial derived progenitor cells (MASCs). Upon differentiation, the latter produced a variety tissues including functional endothelium, illustrating the potential applications of such cells. Thus, GPR125 represents a novel target for purifying adult stem and progenitors from tissues, with the goal of developing autologous multipotent cell lines.

Generation of functional multipotent adult stem cells from GPR125+ germline progenitors.

Adult mammalian testis is a source of pluripotent stem cells. However, the lack of specific surface markers has hampered identification and tracking of the unrecognized subset of germ cells that gives rise to multipotent cells. Although embryonic-like cells can be derived from adult testis cultures after only several weeks in vitro, it is not known whether adult self-renewing spermatogonia in long-term culture can generate such stem cells as well. Here, we show that highly proliferative adult spermatogonial progenitor cells (SPCs) can be efficiently obtained by cultivation on mitotically inactivated testicular feeders containing CD34+ stromal cells. SPCs exhibit testicular repopulating activity in vivo and maintain the ability in long-term culture to give rise to multipotent adult spermatogonial-derived stem cells (MASCs). Furthermore, both SPCs and MASCs express GPR125, an orphan adhesion-type G-protein-coupled receptor. In knock-in mice bearing a GPR125-beta-galactosidase (LacZ) fusion protein under control of the native Gpr125 promoter (GPR125-LacZ), expression in the testis was detected exclusively in spermatogonia and not in differentiated germ cells. Primary GPR125-LacZ SPC lines retained GPR125 expression, underwent clonal expansion, maintained the phenotype of germline stem cells, and reconstituted spermatogenesis in busulphan-treated mice. Long-term cultures of GPR125+ SPCs (GSPCs) also converted into GPR125+ MASC colonies. GPR125+ MASCs generated derivatives of the three germ layers and contributed to chimaeric embryos, with concomitant downregulation of GPR125 during differentiation into GPR125- cells. MASCs also differentiated into contractile cardiac tissue in vitro and formed functional blood vessels in vivo. Molecular bookmarking by GPR125 in the adult mouse and, ultimately, in the human testis could enrich for a population of SPCs for derivation of GPR125+ MASCs, which may be employed for genetic manipulation, tissue regeneration and revascularization of ischaemic organs.

Cytokine-mediated deployment of SDF-1 induces revascularization through recruitment of CXCR4+ hemangiocytes.

The mechanisms through which hematopoietic cytokines accelerate revascularization are unknown. Here, we show that the magnitude of cytokine-mediated release of SDF-1 from platelets and the recruitment of nonendothelial CXCR4+ VEGFR1+ hematopoietic progenitors, 'hemangiocytes,' constitute the major determinant of revascularization. Soluble Kit-ligand (sKitL), thrombopoietin (TPO, encoded by Thpo) and, to a lesser extent, erythropoietin (EPO) and granulocyte-macrophage colony-stimulating factor (GM-CSF) induced the release of SDF-1 from platelets, enhancing neovascularization through mobilization of CXCR4+ VEGFR1+ hemangiocytes. Although revascularization of ischemic hindlimbs was partially diminished in mice deficient in both GM-CSF and G-CSF (Csf2-/- Csf3-/-), profound impairment in neovascularization was detected in sKitL-deficient Mmp9-/- as well as thrombocytopenic Thpo-/- and TPO receptor-deficient (Mpl-/-) mice. SDF-1-mediated mobilization and incorporation of hemangiocytes into ischemic limbs were impaired in Thpo-/-, Mpl-/- and Mmp9-/- mice. Transplantation of CXCR4+ VEGFR1+ hemangiocytes into Mmp9-/- mice restored revascularization, whereas inhibition of CXCR4 abrogated cytokine- and VEGF-A-mediated mobilization of CXCR4+ VEGFR1+ cells and suppressed angiogenesis. In conclusion, hematopoietic cytokines, through graded deployment of SDF-1 from platelets, support mobilization and recruitment of CXCR4+ VEGFR1+ hemangiocytes, whereas VEGFR1 is essential for their angiogenic competency for augmenting revascularization. Delivery of SDF-1 may be effective in restoring angiogenesis in individuals with vasculopathies.

VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche.

The cellular and molecular mechanisms by which a tumour cell undergoes metastasis to a predetermined location are largely unknown. Here we demonstrate that bone marrow-derived haematopoietic progenitor cells that express vascular endothelial growth factor receptor 1 (VEGFR1; also known as Flt1) home to tumour-specific pre-metastatic sites and form cellular clusters before the arrival of tumour cells. Preventing VEGFR1 function using antibodies or by the removal of VEGFR1(+) cells from the bone marrow of wild-type mice abrogates the formation of these pre-metastatic clusters and prevents tumour metastasis, whereas reconstitution with selected Id3 (inhibitor of differentiation 3)-competent VEGFR1+ cells establishes cluster formation and tumour metastasis in Id3 knockout mice. We also show that VEGFR1+ cells express VLA-4 (also known as integrin alpha4beta1), and that tumour-specific growth factors upregulate fibronectin--a VLA-4 ligand--in resident fibroblasts, providing a permissive niche for incoming tumour cells. Conditioned media obtained from distinct tumour types with unique patterns of metastatic spread redirected fibronectin expression and cluster formation, thereby transforming the metastatic profile. These findings demonstrate a requirement for VEGFR1+ haematopoietic progenitors in the regulation of metastasis, and suggest that expression patterns of fibronectin and VEGFR1+VLA-4+ clusters dictate organ-specific tumour spread.

Tie2 activation contributes to hemangiogenic regeneration after myelosuppression.

Chemotherapy- or radiation-induced myelosuppression results in apoptosis of cycling hematopoietic cells and induces regression of bone marrow (BM) sinusoidal vessels. Moreover, timely regeneration of BM neovessels is essential for reconstitution of hematopoiesis. However, the identity of angiogenic factors that support reconstitution of BM's vasculature is unknown. Here, we demonstrate that angiopoietin/tyrosine kinase with immunoglobulin and epidermal growth factor homology domains-2 (Tie2) signaling contributes to the assembly and remodeling of BM neovessels after myelosuppression. Using transgenic mice where the Tie2 promoter drives the reporter LacZ gene (Tie2-LacZ), we demonstrate that at steady state, there was minimal expression of Tie2 in the BM vasculature. However, after 5-fluorouracil (5-FU) treatment, there was a rapid increase in plasma vascular endothelial growth factor A (VEGF-A) levels and expansion of Tie2-positive neovessels. Inhibition of Tie2 resulted in impaired neoangiogenesis, leading to a delay in hematopoietic recovery. Conversely, angiopoietin-1 (Ang-1) stimulated hematopoiesis both in wild-type and thrombopoietin-deficient mice. In addition, Ang-1 shortened the duration of chemotherapy-induced neutropenia in wild-type mice. Exogenous VEGF-A and Ang-1 stimulated Tie2 expression in the BM vasculature. These data suggest that VEGF-A-induced up-regulation of Tie2 expression on the regenerating vasculature after BM suppression supports the assembly of sinusoidal endothelial cells, thereby promoting reconstitution of hematopoiesis. Angiopoietins may be clinically useful to accelerate hemangiogenic recovery after myelosuppression.

Cytokine preconditioning promotes codifferentiation of human fetal liver CD133+ stem cells into angiomyogenic tissue.

BACKGROUND: CD133 (AC133) is a surface antigen that defines a broad population of stem cells, including myogenic and endothelial progenitors. CD133+ cells are rare in adult tissues, and the factors that support their differentiation into mature angiomyogenic cells are not known. These hurdles have hampered the use of CD133+ cells for therapeutic purposes. Because human fetal liver is a rich source of CD133+ cells, we sought to identify the growth factors that promote codifferentiation of these cells into angiogenic and myogenic cells. METHODS AND RESULTS: Human fetal liver CD133+ and CD133- cell subpopulations were cultured with 5'-azacytidine or vascular endothelial growth factor (VEGF165) and/or brain-derived nerve growth factor (BDNF). CD133+ but not CD133- cells from human fetal liver codifferentiated into spindle-shaped cells, as well as flat adherent multinucleated cells capable of spontaneous contractions in culture. The resulting spindle-shaped cells were confirmed to be endothelial cells by immunohistochemistry analysis for von Willebrand factor and by acetylated LDL uptake. Multinucleated cells were characterized as striated muscles by electron microscopy and immunohistochemistry analysis for myosin heavy chain. Presence of VEGF165 and BDNF significantly enhanced angiomyogenesis in vitro. Inoculation of cells derived from CD133+ cells, but not CD133- cells, into the ear pinna of NOD/SCID mice resulted in the formation of cardiomyocytes, as identified by immunostaining with cardiac troponin-T antibody. These cells generated electrical action potentials, detectable by ECG tracing. CONCLUSIONS: CD133 defines a population of human fetal liver cells capable of differentiating into both angiogenic and myogenic cells. Preconditioning of these CD133+ cells with VEGF165 and BDNF enhances the angiomyogenesis. CD133+ fetal liver cells ultimately may be used for therapeutic angiomyogenesis.

AC133/CD133/Prominin-1.

Prominin-1, originally found on neuroepithelial stem cells in mice, is a five transmembrane domain cell-surface glycoprotein that localizes to membrane protrusions. Its homologue human Prominin-1 was first isolated from hematopoietic stem cells by a monoclonal antibody recognizing a specific epitope designated as AC133 (CD133). Transcription of Prominin-1 is driven by five tissue-specific alternative promoters resulting in the formation of differentially spliced mRNA isoforms. Prominin-1 is expressed on different types of stem cells, but it is not known if it plays a significant role in key stem cell functional features. Although the biological function of Prominin-1 is not well understood, the AC133 epitope currently serves as a useful marker for the isolation of hematopoietic and endothelial progenitor cells.

Alternative promoters regulate transcription of the gene that encodes stem cell surface protein AC133.

AC133 is a member of a novel family of cell surface proteins with 5 transmembrane domains. The function of AC133 is unknown. Although AC133 mRNA is detected in different tissues, its expression in the hematopoietic system is restricted to CD34+ stem cells. AC133 is also expressed on stem cells of other tissues, including endothelial progenitor cells. However, despite the potential importance of AC133 to the field of stem cell biology, nothing is known about the transcriptional regulation of AC133 expression. In this report we showed that the human AC133 gene has at least 9 distinctive 5'-untranslated region (UTR) exons, resulting in the formation of at least 7 alternatively spliced 5'-UTR isoforms of AC133 mRNA, which are expressed in a tissue-dependent manner. We found that transcription of these AC133 isoforms is controlled by 5 alternative promoters, and we demonstrated their activity on AC133-expressing cell lines using a luciferase reporter system. We also showed that in vitro methylation of 2 of these AC133 promoters completely suppresses their activity, suggesting that methylation plays a role in their regulation. Identification of tissue-specific AC133 promoters may provide a novel method to isolate tissue-specific stem and progenitor cells.

Green fluorescent protein selectively induces HSP70-mediated up-regulation of COX-2 expression in endothelial cells.

Reporter genes, including green fluorescent protein (GFP), have been used to monitor the expression of transgenes introduced into vascular cells by gene transfer vectors. Here, we demonstrate that GFP by itself can selectively induce expression of certain genes in endothelial cells. Elevation of the cytoplasmic concentration of GFP in endothelial cells, specifically, resulted in a robust upregulation of heat shock protein 70 (HSP70). GFP induced both mRNA and protein expression of HSP70 in a dose-dependent manner. GFP-mediated up-regulation of HSP70 resulted in induction of cyclooxygenase-2 (COX-2) followed by prostaglandin E2 (PGE2) production. GFP-mediated up-regulation of HSP70 is independent of mitogen-activated protein kinase and phosphatidylinositol-3-kinase signaling cascades because inhibition of these pathways had no effect on HSP70 increases. Adenoviral delivery of GFP into murine vasculature significantly enhanced blood flow, suggesting that sufficient PGE2 is produced to induce vasodilation. Identification of the molecular partners that interact with GFP will increase our understanding of the vascular-specific factors that regulate stress angiogenesis and hemostasis.

VEGF(165) promotes survival of leukemic cells by Hsp90-mediated induction of Bcl-2 expression and apoptosis inhibition.

Similar to endothelial cells (ECs), vascular endothelial growth factor (VEGF) induces Bcl-2 expression on VEGF receptor-positive (VEGFR(+)) primary leukemias and cell lines, promoting survival. We investigated the molecular pathways activated by VEGF on such leukemias, by performing a gene expression analysis of VEGF-treated and untreated HL-60 leukemic cells. One gene to increase after VEGF stimulation was heat shock protein 90 (Hsp90). This was subsequently confirmed at the protein level, on primary leukemias and leukemic cell lines. VEGF increased the expression of Hsp90 by interacting with KDR and activating the mitogen-activated protein kinase cascade. In turn, Hsp90 modulated Bcl-2 expression, as shown by a complete blockage of VEGF-induced Bcl-2 expression and binding to Hsp90 by the Hsp90-specific inhibitor geldanamycin (GA). GA also blocked the VEGF-induced Hsp90 binding to APAF-1 on leukemic cells, a mechanism shown to inhibit apoptosis. Notably, VEGF blocked the proapoptotic effects of GA, correlating with its effects at the molecular level. Earlier, we showed that in some leukemias, a VEGF/KDR autocrine loop is essential for cell survival, whereas here we identified the molecular correlates for such an effect. We also demonstrate that the generation of a VEGF/VEGFR autocrine loop on VEGFR(+) cells such as ECs, also protected them from apoptosis. Infection of ECs with adenovirus-expressing VEGF resulted in elevated Hsp90 levels, increased Bcl-2 expression, and resistance to serum-free or GA-induced apoptosis. In summary, we demonstrate that Hsp90 mediates antiapoptotic and survival-promoting effects of VEGF, which may contribute to the survival advantage of VEGFR(+) cells such as subsets of leukemias.

Identification of differentially expressed genes in sorted cell populations by two-dimensional gene-expression fingerprinting.

Differential activity of genes is one of the major mechanisms underlying a vast array of biological phenomena. Classical genetic approaches (from phenotypes to genes) have proven their exquisite potential for dissection of complex signaling pathways regulating the development of organisms and the functioning of individual cells. In recent years, with the advent of a number of techniques for studying gene function, the reverse genetics approach (from genes to phenotypes) has received broad acceptance. One of the advantages of this strategy is that it makes genes, whose dysfunction either does not produce an evident phenotype or is lethal, amenable to analysis. Reverse genetics has spearheaded the development of procedures for identification of candidate genes for this type of analysis by detecting spatial or temporal changes in geneexpression patterns. A significant range of methods have been proposed (1-7); in particular, the advent of microarray hybridization techniques promises to increase gene-expression analysis throughput by two or more orders of magnitude (8,9). Some of these procedures have been used to identify genes expressed differentially during hematopoiesis (10,11).