Effects of Acute Confinement Stress-induced Hypothalamic-Pituitary Adrenal Axis Activation and Concomitant Peripheral and Central Transforming Growth Factor-ß1 Measures in Nonhuman Primates.

Transforming growth factor-ß1 (TGF-ß1) is a multifunctional cytokine with anti-inflammatory, immunosuppressive and neuroprotective properties. The hypothalamic-pituitary-adrenal (HPA) axis and immune system exert bidirectional influences on each other, via cortisol and TGF-ß1, but the exact nature of the interaction is not well characterized. The current study examined the effects, in bonnet macaques (Macaca radiata), of two consecutive acute confinement stress periods in an unfamiliar room while mildly restrained, first without and then with dexamethasone pretreatment (0.01 mg/kg IM). Preceding the confinement studies, a non-stress control condition obtained contemporaneous levels of cortisol and TGF-ß1 in both plasma and cerebrospinal fluid (CSF) to match the confinement stress studies. Subjects were reared under either normative or variable foraging demand (VFD) conditions. Since there were no rearing effects at baseline or for any of the conditions tested -- either for cortisol or TGF-ß -- the study analyses were conducted on the combined rearing groups. The stress condition increased both plasma and CSF cortisol levels whereas dexamethasone pretreatment decreased cortisol concentrations to below baseline levels despite stress. The stress condition decreased TGF-ß1 concentrations only in CSF but not in serum. Together the data suggested that stress-induced reductions of a centrally active neuroprotective cytokine occurs in the face of HPA axis activation, potentially facilitating glucocortoid-induced neurotoxicity. Stress-induced reductions of neuroprotective cytokines prompts exploration of protective measures against glucocorticoid-induced neurotoxicity.

Adult glucocorticoid receptor mRNA expression volatility in response to an acute stressor and juvenile CSF corticotropin-releasing factor: A pilot neurodevelopmental study.

INTRODUCTION: Early life stress (ELS) has been shown to play a role in establishing persistent maladaptive HPA axis modifications that may contribute to the pathogenesis of mood and anxiety disorders. Central glucocorticoid receptor (GR) messenger RNA (mRNA) expression may facilitate (mal)adaptive responsivity to ELS. The role of adult monocytic GR mRNA expression, a putative CNS proxy, during acute stress exposure was explored as well as the ELS marker, juvenile cerebrospinal fluid (CSF) corticotropin-releasing factor. METHODS: Six adult macaques (three of which were exposed to variable foraging demand, a form of ELS) underwent acute restraint. Baseline GR expression and plasma cortisol concentrations were separately measured followed by subsequent measurements following stress completion (t=0min, 4h, 5 days and 7 days). Juvenile CSF CRF concentrations were available in five subjects to determine their developmental association with GR expression in response to stress. RESULTS: As expected acute restraint stress produced a significant increase in plasma cortisol concentrations most robustly observed at 4h post-stress time point. There was a significant juvenile CSF CRF concentration x time interaction in predicting adult GR mRNA expression in response to stress (partial η2=0.80). During acute stress juvenile CRF concentrations negatively predicted GR expression and during recovery, "flipped" to positively predict expression. Juvenile CSF CRF concentrations positively correlated with the volatility of adult GR mRNA expression. CONCLUSIONS: During acute stress, relatively high CSF CRF concentrations are associated with relatively rapid reductions in GR expression. Return to an ambient post-stress state was characterized by a direct relationship, consistent with increased HPA axis restraint in high CRF subjects. An ELS-associated allostatic adaptation suggests relative elevations of juvenile CSF CRF concentration set the stage for a relative hyper-volatility of adult GR mRNA expression in response to acute stress with potential long-term implications for HPA axis regulation.

Relation of baseline plasma MMP-1 levels to long-term all-cause mortality in patients with known or suspected coronary artery disease referred for coronary angiography.

OBJECTIVES: To investigate the long-term prognostic significance of baseline plasma MMP-1 levels in a group of well-characterized male patients with known or suspected coronary artery disease, including those presenting with acute coronary syndrome. BACKGROUND: MMP-1 is an interstitial collagenase that is considered the primary enzyme responsible for collagen degradation. In addition, MMP-1 can lead to platelet activation through the PAR1 pathway that is independent of thrombin. METHODS: Baseline plasma MMP-1 levels were measured in 364 male patients who were referred for coronary angiography and followed prospectively for five years for the development of all-cause mortality. RESULTS: After adjustment for a variety of baseline clinical, angiographic and laboratory parameters, baseline plasma MMP-1 levels (analyzed as a continuous variable) were an independent predictor of all-cause mortality at 5 years (HR, 1.49; 95% CI, 1.23-1.80; P < 0.0001). Furthermore, in 3 additional multivariate models that included a wide variety of contemporary biomarkers with established prognostic efficacy (i.e., ST2, GDF-15, Cystatin C, hs-CRP, Myeloperoxidase, NT-proBNP, TIMP-1, Adiponectin, RDW, hemoglobin, and Erythropoietin), MMP-1 remained an independent predictor of all-cause mortality at 5 years. Similar results were obtained when the analyses were restricted to the subpopulation of patients presenting with acute coronary syndrome. CONCLUSIONS: Elevated levels of MMP-1 are associated with an increased risk of long-term all-cause mortality in patients with known or suspected coronary disease that is independent of a variety of clinical, angiographic, and laboratory variables, including a whole host of contemporary biomarkers with established prognostic efficacy representing multiple different pathophysiologic processes.

Antimyeloma Effects of the Heat Shock Protein 70 Molecular Chaperone Inhibitor MAL3-101.

Multiple myeloma (MM) is the second most common hematologic malignancy and remains incurable, primarily due to the treatment-refractory/resistant nature of the disease. A rational approach to this compelling challenge is to develop new drugs that act synergistically with existing effective agents. This approach will reduce drug concentrations, avoid treatment resistance, and also improve treatment effectiveness by targeting new and nonredundant pathways in MM. Toward this goal, we examined the antimyeloma effects of MAL3-101, a member of a new class of non-ATP-site inhibitors of the heat shock protein (Hsp) 70 molecular chaperone. We discovered that MAL3-101 exhibited antimyeloma effects on MM cell lines in vitro and in vivo in a xenograft plasmacytoma model, as well as on primary tumor cells and bone marrow endothelial cells from myeloma patients. In combination with a proteasome inhibitor, MAL3-101 significantly potentiated the in vitro and in vivo antimyeloma effects. These data support a preclinical rationale for small molecule inhibition of Hsp70 function, either alone or in combination with other agents, as an effective therapeutic strategy for MM.

Zoledronic acid markedly improves bone mineral density for patients with monoclonal gammopathy of undetermined significance and bone loss.

PURPOSE: Patients with monoclonal gammopathy of undetermined significance (MGUS) have increased rates of bone resorption, osteopenia, osteoporosis, and risk of fractures. This study was undertaken to determine the efficacy and safety of zoledronic acid for patients with MGUS and enhanced bone loss. EXPERIMENTAL DESIGN: In this phase II open-label study, 54 patients with MGUS and osteopenia or osteoporosis were administered zoledronic acid 4 mg i.v. at 0, 6, and 12 months. The primary efficacy end point was bone mineral density, assessed using a dual-energy X-ray absorptiometry scan in the lumbar (L)-spine done at screening and at 13 months (1 month after the final zoledronic acid infusion). RESULTS: At study end for all patients (N = 54), L-spine T-scores improved by a median of +0.27 (range, -0.38 to +3.91), corresponding to a median increase in bone mineral density of +15.0% (range, -18.0% to +1,140.0%; P < 0.0001). Hip T-scores improved by a median of +0.10 (range, -2.40 to +2.03), corresponding to a median increase of +6.0% (range, -350.0% to +165.0%). During the study, no new fractures, osteonecrosis of the jaw, or significant renal adverse events were reported. CONCLUSIONS: Zoledronic acid administered i.v. at a dosage of 4 mg every 6 months for three doses total was well-tolerated and substantially improved bone mineral density for patients with MGUS and bone loss. Zoledronic acid may be effective for the prevention of new fractures in this high-risk population.

Endothelial progenitor cells display clonal restriction in multiple myeloma.

BACKGROUND: In multiple myeloma (MM), increased neoangiogenesis contributes to tumor growth and disease progression. Increased levels of endothelial progenitor cells (EPCs) contribute to neoangiogenesis in MM, and, importantly, covary with disease activity and response to treatment. In order to understand the mechanisms responsible for increased EPC levels and neoangiogenic function in MM, we investigated whether these cells were clonal by determining X-chromosome inactivation (XCI) patterns in female patients by a human androgen receptor assay (HUMARA). In addition, EPCs and bone marrow cells were studied for the presence of clonotypic immunoglobulin heavy-chain (IGH) gene rearrangement, which indicates clonality in B cells; thus, its presence in EPCs would indicate a close genetic link between tumor cells in MM and endothelial cells that provide tumor neovascularization. METHODS: A total of twenty-three consecutive patients who had not received chemotherapy were studied. Screening in 18 patients found that 11 displayed allelic AR in peripheral blood mononuclear cells, and these patients were further studied for XCI patterns in EPCs and hair root cells by HUMARA. In 2 patients whose EPCs were clonal by HUMARA, and in an additional 5 new patients, EPCs were studied for IGH gene rearrangement using PCR with family-specific primers for IGH variable genes (VH). RESULTS: In 11 patients, analysis of EPCs by HUMARA revealed significant skewing (> or = 77% expression of a single allele) in 64% (n = 7). In 4 of these patients, XCI skewing was extreme (> or = 90% expression of a single allele). In contrast, XCI in hair root cells was random. Furthermore, PCR amplification with VH primers resulted in amplification of the same product in EPCs and bone marrow cells in 71% (n = 5) of 7 patients, while no IGH rearrangement was found in EPCs from healthy controls. In addition, in patients with XCI skewing in EPCs, advanced age was associated with poorer clinical status, unlike patients whose EPCs had random XCI. CONCLUSION: Our results suggest that EPCs in at least a substantial subpopulation of MM patients are related to the neoplastic clone and that this is an important mechanism for upregulation of tumor neovascularization in MM.

Circulating endothelial progenitor cells in multiple myeloma: implications and significance.

Angiogenesis governs the progression of multiple myeloma (MM). Circulating endothelial cells (CECs) contribute to angiogenesis and comprise mature ECs and endothelial progenitor cells (EPCs). The present study sought to characterize CECs and their relation to disease activity and therapeutic response in 31 consecutive patients with MM. CECs, identified as CD34(+)/CD146(+)/CD105(+)/CD11b(-) cells, were 6-fold higher in patients compared to controls and correlated positively with serum M protein and beta(2)-microglobulin. Circulating EPCs displayed late colony formation/outgrowth and capillary-like network formation on matrigel; these processes were inhibited after effective thalidomide treatment. Co-expression of vascular endothelial growth factor receptor-2 (KDR) and CD133 characterized EPCs in MM, and KDR mRNA elevations correlated with M protein levels. In vitro exposure of ECs to thalidomide or its derivative CC-5013 inhibited gene expression of the receptors for transforming growth factor-beta and thrombin. Thus, elevated levels of CECs and EPCs covary with disease activity and response to thalidomide, underscoring the angiogenic aspect of MM and suggesting that angioblastlike EPCs are a pathogenic biomarker and a rational treatment target in MM. The results also highlight the anti-angiogenic properties of thalidomide and CC-5013 and further elucidate possible mechanisms of their effectiveness against MM. (Blood. 2005;105:3286-3294).

Regulation of MCP-1 gene transcription by Smads and HIV-1 Tat in human glial cells.

Expression of several cytokines involved in signal transduction such as TGFbeta-1 and the inflammatory chemokines including MCP-1 is elevated during the course of AIDS progression. The enhancement of these cellular proteins in astrocytic cells is mediated, at least in part, by HIV-1 Tat protein. Here, we investigate the possible regulation of MCP-1 transcription by Tat and the Smad family of transcription factors whose activities are induced by the TGFbeta-1 pathway. Results from transfection studies revealed that Smad-3 stimulates basal and Tat-mediated transcription of MCP-1 in human astrocytic cells. Smad-4, on the other hand, had no effect on the basal activity of the MCP-1 promoter, but showed the ability to decrease both Smad-3 and Tat-induced transcription of the MCP promoter. Results from protein-binding studies revealed the ability of both Smad-3 and Smad-4 to associate with the region of Tat spanning residues 1-40. Examination of the transcriptional activity of the various domains of Smad including MH1, at the N-terminus, and MH2, at the C-terminus of the protein indicated that neither MH1 or MH2 alone positively cooperate with Tat in modulating MCP-1 transcription. However, ectopic expression of MH1 and, more notably, MH2 severely suppressed transcriptional activation of MCP-1 by Tat in astrocytic cells. Binding studies revealed that similar to the full-length Smad protein, both MH1 and MH2 associate with Tat protein and that the residues between 1 and 40 of Tat are important for their interaction. These observations reveal a novel mechanism for Tat-mediated transcriptional activation via TGFbeta signaling pathway and provide evidence for regulation of MCP-1 gene transcription by this signaling pathway in human astrocytic cells.

Cellular response to hypoxia involves signaling via Smad proteins.

The transforming growth factor-beta (TGF-beta) family of cytokines regulates vascular development and inflammatory responses. We have recently shown that exposure of human umbilical vein endothelial cells (HUVECs) to hypoxia (1% O(2)) increases gene expression and bioactivation of TGF-beta2 and induces its downstream effectors, Smad proteins (Smads), to associate with DNA. In the present study, we show that hypoxia-induced TGF-beta2 gene expression is dependent on thrombospondin-1-mediated bioactivation of latent TGF-beta. Blocking TGF-beta2 but not TGF-beta1 in hypoxic endothelial cell cultures inhibited induction of the TGF-beta2 gene, indicating that an autocrine mechanism driven by bioactivation of TGF-beta2 leads to its gene expression in hypoxic HUVECs. Exposure of HUVECs to hypoxia resulted in phosphorylation and nuclear transportation of Smad2 and Smad3 proteins as well as stimulation of transcriptional activities of Smad3 and the transcription factor hypoxia-inducible factor-1alpha and culminated in up-regulation of TGF-beta2 gene expression. Autocrine regulation of TGF-beta2 production in hypoxia may involve cross-talk between Smad3 and HIF-1alpha signaling pathways, and could be an important mechanism by which endothelial cells respond to hypoxic stress.

F11-receptor (F11R/JAM) mediates platelet adhesion to endothelial cells: role in inflammatory thrombosis.

The F11 receptor (F11R) is a cell adhesion molecule (CAM), member of the immunoglobulin superfamily found on the surface of human platelets, and determined to play a role in platelet aggregation, secretion, adhesion and spreading. The same molecule is present also at tight junctions of endothelial cells (EC) where it is known as JAM and acts as a CAM through homophilic interactions. The role of F11R/JAM in the interaction of platelets with endothelial cells was investigated in the current studies. We report here that washed human platelets adhere specifically to a matrix made of immobilized, recombinant sF11R. Furthermore, platelets adhere to cytokine- (TNF-alpha, INF-gamma) stimulated human umbilical vein endothelial cells (HUVEC), and approximately 40-60% of the adhesive force is exerted by homophilic interactions between the F11R of platelets and EC. This is evidenced by the inhibition of platelet adhesion to endothelial cells by recombinant soluble form of the F11R, and by two F11R peptides with amino acid sequences of the N-terminal region, and in the 1(st) Ig fold of the F11R, respectively. This study suggests a role for F11R in the adhesion of platelets to cytokine-inflamed endothelial cells and thus in thrombosis and atherosclerosis induced in non-denuded blood vessels by inflammatory processes. Agents that block the F11R-mediated adhesion of platelets to EC may be of therapeutic value in controlling thrombosis and preventing heart attacks and stroke.

Interaction between TGFbeta signaling proteins and C/EBP controls basal and Tat-mediated transcription of HIV-1 LTR in astrocytes.

Signal transduction pathways induced by cytokines can modulate the level of HIV-1 gene transcription and replication in a variety of cells including those from the central nervous system. Here, we investigated the effect of TGFbeta-1 signaling the factors, including Smads, on transcription of the viral LTR in human astrocytic cells. Ectopic expression of Smad-3 increased activity of the viral promoter, while its partner protein, Smad-4, caused a slight decrease in viral gene transcription. Further, Smad-4 was able to suppress transcriptional activation of the LTR by Smad-3 as well as by C/EBPbeta, another activator of LTR transcription in these cells. Results from promoter deletion experiments identified the C/EBP-binding site, which is positioned between nucleotides -114 and -102 as one of the targets for Smad-mediated regulation of the LTR. Band-shift studies showed inhibition of C/EBP binding to its target DNA in protein extract from cells overexpressing Smad-3 and Smad-4. Results from GST pull-down assay and combined immunoprecipitation/Western blot of protein extracts from human astrocytes verified the association of Smad-3 and Smad-4 with C/EBPbeta, suggesting that interaction of C/EBPbeta with Smad-3 and Smad-4 may have a negative impact upon C/EBPbeta-mediated activation of the LTR. Interestingly, Smad-4 showed no inhibitory effect on viral gene transcription in cells expressing Tat protein. However, in the presence of Smad-3, expression of Smad-4 exerted a negative effect on Tat-mediated activation of the LTR promoter. These observations pointed to the functional interplay between viral and cellular proteins in modulating LTR transcription.

Differential, tissue-specific, transcriptional regulation of apolipoprotein B secretion by transforming growth factor beta.

Apolipoprotein B (apoB) is required for the assembly and secretion of triglyceride-rich lipoproteins. ApoB synthesis is constitutive, and post-translational mechanisms modulate its secretion. Transforming growth factor beta (TGF-beta) increased apoB secretion in both differentiated and nondifferentiated Caco-2 cells and decreased secretion in HepG2 cells without affecting apolipoprotein A-I secretion. TGF-beta altered apoB secretion by changing steady-state mRNA levels and protein synthesis. Expression of SMAD3 and SMAD4 differentially regulated apoB secretion in these cells. Thus, SMADs mediate dissimilar secretion of apoB in both the cell lines by affecting gene transcription. We identified a 485-bp element, 55 kb upstream of the apob gene that contains a SMAD binding motif. This motif increased the expression of chloramphenicol acetyltransferase in Caco-2 cells treated with TGF-beta or transfected with SMADs. Hence, TGF-beta activates SMADs that bind to the 485-bp intestinal enhancer element in the apob gene and increase its transcription and secretion in Caco-2 cells. This is the first example showing differential transcriptional regulation of the apob gene by cytokines and dissimilar regulation of one gene in two different cell lines by TGF-beta. In this regulation, the presence of cytokine-responsive motif in the tissue-specific enhancer element confers cell-specific response.

Transforming growth factor-beta 1 and cortisol in differentially reared primates.

Exposure of primate infants to adverse rearing conditions during the first half year of life can result in enduring behavioral, neuroendocrine, and immunologic abnormalities. However, the effects of differential rearing on cytokines, some of which can regulate immune and inflammatory responses and modulate activity of the central nervous system and the hypothalamic-pituitary-adrenal (HPA) axis, are largely unexamined. The present study explored the relationship between circulating levels of transforming growth factor-beta 1 (TGF-beta 1) and cortisol in macaques reared either normally or under conditions of variable foraging demand (VFD). Under VFD rearing, for a period of 4 months, the infants' mothers intermittently had to expend more time and effort to obtain food than did the mothers of normally reared control subjects. Two years after cessation of the rearing experience, exposure to a moderate stressor (confinement in an unfamiliar room for 90 min) induced elevated levels of serum TGF-beta 1 and plasma cortisol in VFD-reared monkeys compared to normally reared controls. The correlation between TGF-beta 1 and cortisol levels was substantially higher in the normally reared subjects. Examination of the relationship between HPA axis and immune function will improve our understanding of the pathophysiological consequences of adverse rearing.