Mechanism of erythropoietin regulation by angiotensin II.

Erythropoietin (EPO) is the primary regulator of red blood cell development. Although hypoxic regulation of EPO has been extensively studied, the mechanism(s) for basal regulation of EPO are not well understood. In vivo studies in healthy human volunteers and animal models indicated that angiotensin II (Ang II) and angiotensin converting enzyme inhibitors regulated blood EPO levels. In the current study, we found that Ang II induced EPO expression in situ in murine kidney slices and in 786-O kidney cells in culture as determined by reverse transcription polymerase chain reaction. We further investigated the signaling mechanism of Ang II regulation of EPO in 786-O cells. Pharmacological inhibitors of Ang II type 1 receptor (AT1R) and extracellular signal-regulated kinase 1/2 (ERK1/2) suppressed Ang II transcriptional activation of EPO. Inhibitors of AT2R or Src homology 2 domain-containing tyrosine phosphatase had no effect. Coimmunoprecipiation experiments demonstrated that p21Ras was constitutively bound to the AT1R; this association was increased by Ang II but was reduced by the AT1R inhibitor telmisartan. Transmembrane domain (TM) 2 of AT1R is important for G protein-dependent ERK1/2 activation, and mutant D74E in TM2 blocked Ang II activation of ERK1/2. Ang II signaling induced the nuclear translocation of the Egr-1 transcription factor, and overexpression of dominant-negative Egr-1 blocked EPO promoter activation by Ang II. These data identify a novel pathway for basal regulation of EPO via AT1R-mediated Egr-1 activation by p21Ras-mitogen-activated protein kinase/ERK kinase-ERK1/2. Our current data suggest that Ang II, in addition to regulating blood volume and pressure, may be a master regulator of erythropoiesis.

Angiotensin-II-induced apoptosis requires regulation of nucleolin and Bcl-xL by SHP-2 in primary lung endothelial cells.

Angiotensin II (Ang II) is a key proapoptotic factor in fibrotic tissue diseases. However, the mechanism of Ang-II-induced cell death in endothelial cells has not been previously elucidated. Using the neutral comet assay and specific receptor antagonists and agonists, we found that Ang-II-mediated apoptosis in primary pulmonary endothelial cells required the AT2 receptor. Ang II caused cytochrome c release from the mitochondria concurrent with caspase-3 activation and DNA fragmentation, and apoptosis was suppressed by an inhibitor of Bax-protein channel formation, implicating mitochondrial-mediated apoptosis. There was no evidence that the extrinsic apoptotic pathway was involved, because caspase-9, but not caspase-8, was activated by Ang-II treatment. Apoptosis required phosphoprotein phosphatase activation, and inhibition of the SHP-2 phosphatase (encoded by Ptpn11) blocked cell death. Reduced levels of anti-apoptotic Bcl-2-family members can initiate intrinsic apoptosis, and we found that Ang-II treatment lowered cytosolic Bcl-x(L) protein levels. Because the protein nucleolin has been demonstrated to bind Bcl-x(L) mRNA and prevent its degradation, we investigated the role of nucleolin in Ang-II-induced loss of Bcl-x(L). RNA-immunoprecipitation experiments revealed that Ang II reduced the binding of nucleolin to Bcl-x(L) mRNA in an AU-rich region implicated in instability of Bcl-x(L) mRNA. Inhibition of SHP-2 prevented Ang-II-induced degradation of Bcl-x(L) mRNA. Taken together, our findings suggest that nucleolin is a primary target of Ang-II signaling, and that Ang-II-activated SHP-2 inhibits nucleolin binding to Bcl-x(L) mRNA, thus affecting the equilibrium between pro- and anti-apoptotic members of the Bcl-2 family.

Captopril reduces lung inflammation and accelerated senescence in response to thoracic radiation in mice.

The lung is sensitive to radiation and exhibits several phases of injury, with an initial phase of radiation-induced pneumonitis followed by delayed and irreversible fibrosis. The angiotensin-converting enzyme inhibitor captopril has been demonstrated to mitigate radiation lung injury and to improve survival in animal models of thoracic irradiation, but the mechanism remains poorly understood. Here we investigated the effect of captopril on early inflammatory events in the lung in female CBA/J mice exposed to thoracic X-ray irradiation of 17-17.9 Gy (0.5-0.745 Gy min-1). For whole-body + thoracic irradiation, mice were exposed to 7.5 Gy (0.6 Gy min-1) total-body 60Co irradiation and 9.5 Gy thoracic irradiation. Captopril was administered orally (110 mg kg-1 day-1) in the drinking water, initiated 4 h through to150 days post-irradiation. Captopril treatment increased survival from thoracic irradiation to 75% at 150 days compared with 0% survival in vehicle-treated animals. Survival was characterized by a significant decrease in radiation-induced pneumonitis and fibrosis. Investigation of early inflammatory events showed that captopril significantly attenuated macrophage accumulation and decreased the synthesis of radiation-induced interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) pro-inflammatory cytokines in the lungs of irradiated mice. Suppression of IL-1ß and TNF-α correlated with an increase of the anti-inflammatory cytokine IL-10 in the spleen with captopril treatment. We also found that captopril decreased markers for radiation-induced accelerated senescence in the lung tissue. Our data suggest that suppression of inflammation and senescence markers, combined with an increase of anti-inflammatory factors, are a part of the mechanism for captopril-induced survival in thoracic irradiated mice.

Effects of captopril against radiation injuries in the Göttingen minipig model of hematopoietic-acute radiation syndrome.

Our laboratory has demonstrated that captopril, an angiotensin converting enzyme inhibitor, mitigates hematopoietic injury following total body irradiation in mice. Improved survival in mice is correlated with improved recovery of mature blood cells and bone marrow, reduction of radiation-induced inflammation, and suppression of radiation coagulopathy. Here we investigated the effects of captopril treatment against radiation injuries in the Göttingen mini pig model of Hematopoietic-Acute Radiation Syndrome (H-ARS). Minipigs were given captopril orally (0.96 mg/kg) twice daily for 12 days following total body irradiation (60Co 1.79 Gy, 0.42-0.48 Gy/min). Blood was drawn over a time course following irradiation, and tissue samples were collected at euthanasia (32-35 days post-irradiation). We observed improved survival with captopril treatment, with survival rates of 62.5% in vehicle treated and 87.5% in captopril treated group. Additionally, captopril significantly improved recovery of peripheral blood mononuclear cells, and a trend toward improvement in recovery of red blood cells and platelets. Captopril significantly reduced radiation-induced expression of cytokines erythropoietin and granulocyte-macrophage colony-stimulating factor and suppressed radiation-induced acute-phase inflammatory response cytokine serum amyloid protein A. Using quantitative-RT-PCR to monitor bone marrow recovery, we observed significant suppression of radiation-induced expression of redox stress genes and improved hematopoietic cytokine expression. Our findings suggest that captopril activities in the Göttingen minipig model of hematopoietic-acute radiation syndrome reflect findings in the murine model.

Bleomycin induces the extrinsic apoptotic pathway in pulmonary endothelial cells.

Bleomycin, a chemotherapeutic agent, can cause pulmonary fibrosis in humans and is commonly used to induce experimental pulmonary fibrosis in rodents. In cell culture, bleomycin causes single- and double-stranded DNA breaks and produces reactive oxidative species, both of which require iron (Fe(2+)) and O(2). The mechanism of bleomycin-induced apoptosis is controversial due to its complexity. We investigated bleomycin apoptotic signaling events in primary pulmonary endothelial cells. Time course experiments revealed that bleomycin induced apoptosis within 4 h. Caspase-8, the initiator caspase for the extrinsic pathway, was activated within 2 h and preceded activation of the effector caspases-3 and -6 (4 h). Caspase-9, the initiator of the intrinsic pathway and release of cytochrome c from the mitochondria were not detected at these time points. Bleomycin induced the expression of Bcl-2 and Bcl-x(L), Bcl-2 family member proteins that protect cells from the mitochondria-dependent intrinsic apoptosis. Real-time quantitative RT-PCR results demonstrated that, at 4-8 h, bleomycin induced expression of TNF and TNF receptor family genes known to induce the extrinsic apoptotic pathway. Silencing of the death receptor adaptor protein Fas-associated death domain by short interfering RNA significantly reduced bleomycin-induced apoptosis. Apoptosis was also abrogated by caspase-8 inhibition, but only slightly reduced by caspase-3 inhibition. Together, these data suggest that bleomycin initiates apoptosis via the extrinsic pathway.

A tandem repeat of a fragment of Listeria monocytogenes internalin B protein induces cell survival and proliferation.

Hepatocyte growth factor (HGF) is critical for tissue homeostasis and repair in many organs including the lung, heart, kidney, liver, nervous system, and skin. HGF is a heterodimeric protein containing 20 disulfide bonds distributed among an amino-terminal hairpin, four kringle domains, and a serine protease-like domain. Due to its complex structure, recombinant production of HGF in prokaryotes requires denaturation and refolding, processes that are impractical for large-scale manufacture. Thus, pharmaceutical quantities of HGF are not available despite its potential applications. A fragment of the Listeria monocytogenes internalin B protein from amino acids 36-321 (InlB36₋321) was demonstrated to bind to and partially activate the HGF receptor Met. InlB36₋321 has a stable ß-sheet structure and is easily produced in its native conformation by Escherichia coli. We cloned InlB36₋321 (1×InlB36₋321) and engineered a head-to-tail repeat of InlB36₋321 with a linker peptide (2×InlB36₋321); 1×InlB36₋321 and 2×InlB36₋321 were purified from E. coli. Both 1× and 2×InlB36₋321 activated the Met tyrosine kinase. We subsequently compared signal transduction of the two proteins in primary lung endothelial cells. 2×InlB36₋321 activated ERK1/2, STAT3, and phosphatidylinositol 3-kinase/Akt pathways, whereas 1×InlB36₋321 activated only STAT3 and ERK1/2. The 2×InlB36₋321 promoted improved motility compared with 1×InlB36₋321 and additionally stimulated proliferation equivalent to full-length HGF. Both the 1× and 2×InlB36₋321 prevented apoptosis by the profibrotic peptide angiotensin II in cell culture and ex vivo lung slice cultures. The ease of large-scale production and capacity of 2×InlB36₋321 to mimic HGF make it a potential candidate as a pharmaceutical agent for tissue repair.

Delayed Captopril Administration Mitigates Hematopoietic Injury in a Murine Model of Total Body Irradiation.

The increasing potential for accidental radiation exposure from either nuclear accidents or terrorist activities has escalated the need for radiation countermeasure development. We previously showed that a 30-day course of high-dose captopril, an ACE inhibitor, initiated 1-4 h after total body irradiation (TBI), improved Hematopoietic Acute Radiation Syndrome (H-ARS) and increased survival in mice. However, because of the time likely required for the deployment of a stockpiled radiation countermeasure to a radiation mass casualty site, there is a need for therapies that can be administered 24-48 hours after initial exposure. Using C57BL/6 mice exposed to an LD50-80/30 of 60Co TBI (7.75-7.9 Gy, 0.615 Gy/min), we show that low-dose captopril administration, initiated as late as 48 h post-TBI and continued for 14 days, significantly enhanced overall survival similarly to high-dose, rapid administration. Captopril treatment did not affect radiation-induced cell cycle arrest genes or the immediate loss of hematopoietic precursors. Reduced mortality was associated with the recovery of bone marrow cellularity and mature blood cell recovery at 21-30 days post-irradiation. Captopril reduced radiation-induced cytokines EPO, G-CSF, and SAA in the plasma. Finally, delayed captopril administration mitigated brain micro-hemorrhage at 21 days post-irradiation. These data indicate that low dose captopril administered as late as 48 h post-TBI for only two weeks improves survival that is associated with hematopoietic recovery and reduced inflammatory response. These data suggest that captopril may be an ideal countermeasure to mitigate H-ARS following accidental radiation exposure.

Characterization of the bovine angiotensin converting enzyme promoter: essential roles of Egr-1, ATF-2 and Ets-1 in the regulation by phorbol ester.

The protease angiotensin converting enzyme (ACE) is a key regulator of blood pressure homeostasis, and is responsible for proteolytic activation of angiotensin I to angiotensin II (Ang II), a potent vasoconstrictor, and proteolytic inactivation of bradykinin, a vasodilator. Recent studies have also implicated ACE and Ang II dysregulation in the progression of fibrotic tissue diseases. Although many studies have utilized bovine tissues and cells for investigating the regulation of ACE gene expression, the bovine ACE promoter has not been previously characterized. Here we present the analysis of the bovine ACE promoter. We investigated cis elements regulated by phorbol 12-myristate 13-acetate (PMA). Sequence analysis shows that the bovine ACE promoter contains several putative binding sites for the transcription factors ATF-2, Ets-1, Egr-1 and SP1/SP3. Chromatin immunoprecipitation (ChIP) indicated that the activation of the bovine ACE promoter by PMA involves histone H4 acetylation, and that PMA induced Egr-1 and ATF-2 binding to the ACE promoter, whereas Ets-1 binding was suppressed by PMA. The regulatory roles of these transcription factors in the bovine ACE gene regulation were confirmed by co-expression of either wild type or dominant negative transcription factors with the luciferase reporter constructs. The bovine and human ACE promoters share similarities in binding sites for transcription factors and PMA regulation within the core regions but contain significant differences in the distal promoter regions.

Genistein induces radioprotection by hematopoietic stem cell quiescence.

PURPOSE: In this study we addressed whether genistein-induced radioprotection in mice is associated with alterations of the cell cycle of hematopoietic stem and progenitor cells. MATERIALS AND METHODS: C57BL/6J female mice received a single subcutaneous injection of genistein (200 mg/kg) 24 h prior to a lethal dose (7.75 Gy, (60)Co) of total body irradiation. Proliferation-associated Ki-67 protein/7-aminoactinomycin-D (Ki67/7AAD) cell cycle staining was used to differentiate between G(0), G(1), and S/G(2)/M in bone marrow cell populations negative for expression of mature hematopoietic lineage marker cells but positive for expression of stem cell antigen-1 and tyrosine kinase receptor for stem cell factor (Lin(-)Sca-1(+)cKit(+), LSK(+)). Quantitative real-time polymerase chain reaction (qRT-PCR) microarrays were utilized to examine cell cycle specific genes. RESULTS: At 24 h following radiation exposure, a greater percentage of LSK(+) in genistein-treated mice accumulated in the G(0) phase of the cell cycle, whereas a large percentage of LSK(+) bone marrow cells from untreated and vehicle (PEG-400)-treated mice progressed into the G(1) and S/G(2)/M phases. Moreover, the absolute number of marrow total LSK(+), long-term LSK(+), and short-term LSK(+) increased 2.8, 12.1, and 4.2-fold, respectively, at 7 days post-irradiation in genistein-treated vs. untreated irradiated mice. Lin(-) cells from genistein-treated mice expressed fewer DNA damage responsive and cell cycle checkpoint genes than LSK(+) from untreated or vehicle-treated mice. CONCLUSION: Pretreatment with genistein provides in vivo protection from acute myelotoxicity through extended quiescence followed by reduced senescence of marrow repopulating LSK(+).

Pathogenic and rare deleterious variants in multiple genes suggest oligogenic inheritance in recurrent exertional rhabdomyolysis.

Exertional rhabdomyolysis is a metabolic event characterized by the release of muscle content into the circulation due to exercise-driven breakdown of skeletal muscle. Recurrent exertional rhabdomyolysis has been associated with metabolic myopathies and mitochondrial disorders, a clinically and genetically heterogeneous group of predominantly autosomal recessive, monogenic conditions. Although genetics factors are well recognized in recurrent rhabdomyolysis, the underlying causes and mechanisms of exercise-driven muscle breakdown remain unknown in a substantial number of cases. We present clinical and genetic study results from seven adult male subjects with recurrent exertional rhabdomyolysis. In all subject, whole exome sequencing identified multiple heterozygous variants in genes associated with monogenic metabolic and/or mitochondrial disorders. These variants consisted of known pathogenic and/or new likely pathogenic variants in combination with other rare deleterious alleles. The presence of heterozygous pathogenic and rare deleterious variants in multiple genes suggests an oligogenic inheritance for exertional rhabdomyolysis etiology. Our data imply that exertional rhabdomyolysis can reflect cumulative effects or synergistic interactions of deleterious variants in multiple genes that are likely to compromise muscle metabolism under the stress of exercise.

Multifactorial Origin of Exertional Rhabdomyolysis, Recurrent Hematuria, and Episodic Pain in a Service Member with Sickle Cell Trait.

Individuals with Sickle Cell Trait (SCT), generally considered a benign carrier state of hemoglobin S (HbAS), are thought to be at risk for exertional rhabdomyolysis and hematuria, conditions that can also be caused by various other acquired and inherited factors. We report an SCT positive service member with an exertional rhabdomyolysis event, recurrent hematuria with transient proteinuria, and episodic burning pain in the lower extremities. Clinical and genetic studies revealed the multifactorial nature of his complex phenotype. The service member was taking prescription medications known to be associated with exertional rhabdomyolysis. He carried a pathogenic mutation, NPHS2 p.V260E, reported in nephropathy and a new variant p.R838Q in SCN11A, a gene involved in familial episodic pain syndrome. Results suggest that drug-to-drug interactions coupled with the stress of exercise, coinheritance of HbAS and NPHS2 p.V260E, and p. R838Q in SCN11A contributed to exertional rhabdomyolysis, recurrent hematuria with proteinuria, and episodic pain, respectively. This case underscores the importance of comprehensive clinical and genetic evaluations to identify underlying causes of health complications reported in SCT individuals.

Erythropoietin Regulation by Angiotensin II.

The renin-angiotensin system (RAS) is a key regulator of blood pressure and blood volume homeostasis. The RAS is primarily comprised of the precursor protein angiotensinogen and the two proteases, renin and angiotensin-converting enzyme (ACE). Angiotensin I (Ang I) is derived from angiotensinogen by renin, but appears to have no biological activity. In contrast, angiotensin II (Ang II) that has a variety of biological functions in the cells is converted from Ang I through removal of two-C-terminal residues by ACE. The physiological effects of Ang II are due to Ang II signaling through specific receptor binding, resulting in muscle contraction leading to increased blood pressure and volume. To modulate RAS, three classes of drugs have been developed: (1) renin inhibitors to prevent angiotensinogen conversion to Ang I, (2) ACE inhibitors, to prevent Ang I processing to Ang II and (3) angiotensin receptor blockers, to inhibit Ang II signaling through its receptor. Studies using the RAS inhibitors and Ang II demonstrated that RAS signaling mediates actions of Ang II in the regulation of proliferation and differentiation of specific hematopoietic cell types, especially in the red blood cell lineage. Accumulating evidence indicates that RAS regulates EPO, an essential mediator of red cell production, for human anemia and erythropoiesis in vivo and in vitro. The regulation of EPO expression by Ang II may be responsible for maintaining red blood cell homeostasis. This review highlights the biological roles of RAS for blood cell and EPO homeostasis through Ang II signaling. The molecular mechanism for Ang II-induced EPO production of the cell or tissue type-specific expression is discussed.

The hepatocyte growth factor isoform NK2 activates motogenesis and survival but not proliferation due to lack of Akt activation.

Hepatocyte growth factor (HGF) is a pleiotrophic factor involved in cellular proliferation, migration and morphogenesis. HGF is required for normal tissue and organ development during embryogenesis, but in the adult HGF has been demonstrated to drive normal tissue repair and inhibit fibrotic remodeling. HGF has two naturally occurring human isoforms as a result of alternative splicing, NK1 and NK2. While NK1 has been defined as an agonist for HGF receptor, Met, NK2 is defined as a partial Met antagonist. Furthermore, under conditions of fibrotic remodeling, NK2 is still expressed while full length HGF is suppressed. Furthermore, the mechanism by which NK2 partially signals through Met is not completely understood. Here, we investigated the mitogenic, motogenic, and anti-apoptotic activities of NK2 compared with full length HGF in primary human bronchial epithelial cells (BEpC) and bovine pulmonary artery endothelial cells (PAEC). In human BEpC, NK2 partial activated Met, inducing Met phosphorylation at Y1234/1235 in the tyrosine-kinase domain but not at Y1349 site in the multifunctional docking domain. Partial phosphorylation of Met by NK2 resulted in activation of MAPK and STAT3, but not AKT. This correlated with motogenesis and survival in a MAPK-dependent manner, but not cell proliferation. Overexpression of a constitutively active AKT complemented NK2 signaling, allowing NK2 to induce cell proliferation. These data indicate that NK2 and HGF drive motogenic and anti-apoptotic signaling but only HGF drives cell proliferation by activating AKT-pathway signaling. These results have implications for the biological consequences of differential regulation of the two isoforms under pro-fibrotic conditions.

Hepatocyte Growth Factor Isoforms in Tissue Repair, Cancer, and Fibrotic Remodeling.

Hepatocyte growth factor (HGF), also known as scatter factor (SF), is a pleotropic factor required for normal organ development during embryogenesis. In the adult, basal expression of HGF maintains tissue homeostasis and is up-regulated in response to tissue injury. HGF expression is necessary for the proliferation, migration, and survival of epithelial and endothelial cells involved in tissue repair in a variety of organs, including heart, lung, kidney, liver, brain, and skin. The administration of full length HGF, either as a protein or using exogenous expression methodologies, increases tissue repair in animal models of tissue injury and increases angiogenesis. Full length HGF is comprised of an N-terminal hairpin turn, four kringle domains, and a serine protease-like domain. Several naturally occurring alternatively spliced isoforms of HGF were also identified. The NK1 variant contains the N-terminal hairpin and the first kringle domain, and the NK2 variant extends through the second kringle domain. These alternatively spliced forms of HGF activate the same receptor, MET, but they differ from the full length protein in their cellular activities and their biological functions. Here, we review the species-specific expression of the HGF isoforms, their regulation, the signal transduction pathways they activate, and their biological activities.

Transforming growth factor-ß1 selectively inhibits hepatocyte growth factor expression via a micro-RNA-199-dependent posttranscriptional mechanism.

Hepatocyte growth factor (HGF) is a multipotent endogenous repair factor secreted primarily by mesenchymal cells with effects on cells expressing its receptor, Met. HGF promotes normal tissue regeneration and inhibits fibrotic remodeling in part by promoting proliferation and migration of endothelial and epithelial cells and protecting these cells from apoptosis. HGF also inhibits myofibroblast proliferation. The profibrotic cytokine transforming growth factor beta 1 (TGF-ß1) suppresses HGF expression but not the expression of NK2, an HGF splice variant that antagonizes HGF-induced proliferation. We investigated the mechanism for differential regulation of HGF and NK2 by TGF-ß1. TGF-ß1 down-regulated HGF in primary human adult pulmonary fibroblasts (HLFb) and increased the expression of miR-199a-3p, a microRNA (miRNA) associated with fibrotic remodeling. HGF and NK2 contain completely different 3' untranslated regions (UTRs), and we determined that miR-199a-3p targeted HGF mRNA for suppression but not NK2. A pre-miR-199 mimic inhibited the expression of a luciferase reporter harboring the HGF 3' UTR but not a pmirGLO reporter containing the NK2 3' UTR. In contrast, an anti-miRNA inhibitor specific for miR-199a-3p prevented TGF-ß1-induced reduction of both HGF mRNA and HGF protein secretion. Taken together, these findings demonstrate that HGF is distinctly regulated at the posttranscriptional level from its antagonist NK2.

X-irradiation induces ER stress, apoptosis, and senescence in pulmonary artery endothelial cells.

PURPOSE: The use of clinical radiation for cancer treatment is limited by damage to underlying normal tissue including to the vascular endothelium. We investigated the mechanisms of X-ray-induced cell damage to endothelial cells. METHODS: We evaluated necrosis, apoptosis, cellular senescence, and the contribution of endoplasmic reticulum (ER) stress in pulmonary artery endothelial cells (PAEC) irradiated with X-rays (2-50 Gray [Gy]). RESULTS: Clonogenic assays showed that 10 Gy induced ∼99.9% loss of cell viability. No necrosis was detected using lactate dehydrogenase assays, but a low population underwent extrinsic and intrinsic apoptosis, as indicated by the activation of caspases 3, 8, and 9 as well as by neutral comet assay. A majority of PAEC underwent accelerated senescence, as indicated by morphological changes, increased 21 kD cyclin-dependent kinase inhibitor (p21/waf1), decreased sirtuin 1 (SIRT1), and elevated senescence-associated ß-galactosidase (SA-ß-gal). ER stress was detected by assays for glucose-regulated protein 78 (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP), and growth arrest and DNA damage-inducible protein 34 (GADD34) mRNA, and transient phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α). The ER stress inhibitor salubrinal blocked ∼50% of apoptosis with no effect on senescence. CONCLUSIONS: X-rays primarily induced cellular senescence with limited levels of apoptosis in endothelial cells. ER stress contributed to apoptosis but not to senescence.

Captopril modulates hypoxia-inducible factors and erythropoietin responses in a murine model of total body irradiation.

OBJECTIVE: Our laboratory reported that the angiotensin converting enzyme inhibitor captopril improves erythroid recovery from total body irradiation (TBI) in mice when administered after irradiation. However, captopril administered before TBI attenuates erythroid recovery. Here we investigate captopril and radiation regulation of erythropoietin (EPO) and thrombopoietin (TPO), key effectors of erythroid progenitor proliferation and differentiation. MATERIALS AND METHODS: C57BL/6 mice, nonirradiated or exposed to 7.5 Gy TBI ((60)Co, 0.6 Gy/min) were untreated or administered captopril. Plasma EPO and TPO levels were measured by enzyme-linked immunosorbent assay. Gene expression of EPO was determined by quantitative reverse transcription polymerase chain reaction. The hypoxia-inducible factors (HIF)-1α and -2α were measured by immunoblotting. RESULTS: In nonirradiated mice, continuous captopril administration in the water transiently reduced reticulocytes and red blood cells after 7 and 10 days, respectively. EPO plasma levels and gene expression were reduced below detectable limits after 2 days of captopril treatment, but recovered within 7 days. HIF-1α and HIF-2α were activated preceding reticulocyte and red blood cell recovery. TBI, which ablates early and late-stage erythroid progenitors, activated both HIFs and increased EPO and TPO. Captopril treatment postirradiation suppressed radiation-induced HIF activation and EPO expression. In contrast, captopril administration for 7 days before TBI resulted in earlier EPO induction and activation. Captopril treatment lowered TPO levels in nonirradiated mice, but had minimal effects on radiation-induced TPO. CONCLUSIONS: In nonirradiated mice, captopril biphasically regulates EPO via HIF activation. TBI ablates erythroid progenitors, resulting in hypoxia, HIF activation, and increased EPO expression that are modulated by captopril treatment. These data suggest that short-term suppression of radiation-induced EPO immediately after TBI is favorable for erythroid recovery.

Hepatocyte growth factor inhibits apoptosis by the profibrotic factor angiotensin II via extracellular signal-regulated kinase 1/2 in endothelial cells and tissue explants.

Hepatocyte growth factor (HGF), an endogenous tissue repair factor, attenuates apoptosis in many primary cell types, but the mechanism is not completely understood. Our laboratory demonstrated that angiotensin (Ang) II activates the intrinsic apoptotic pathway in primary endothelial cells (ECs) via reduction of the antiapoptotic protein Bcl-x(L). Ang II decreased Bcl-x(L) mRNA half-life by reducing its binding to nucleolin, a protein that normally binds a 3' AU-rich region and stabilizes Bcl-x(L) mRNA. We hypothesized HGF may block apoptosis induced by Ang II. We used primary EC and ex vivo cultures of rat lung tissue to investigate HGF inhibition of Ang II-induced apoptosis. Our data indicated HGF abrogated Ang II-induced apoptosis by inhibiting cytochrome c release, caspase-3 activation, and DNA fragmentation. RNA-immunoprecipitation experiments demonstrated that HGF stabilized Bcl-x(L) mRNA by increasing nucleolin binding to the 3'-untranslated region that was associated with cytoplasmic localization of nucleolin. Cytoplasmic localization of nucleolin and Bcl-x(L) mRNA stabilization required HGF activation of extracellular signal-regulated kinase (ERK)1/2, but not phosphatidylinositol 3-kinase. HGF also blocked Ang II-induced caspase-3 activation and lactate dehydrogenase release in tissue explants in an ERK-dependent manner.

Gene expression analysis of TFII-I modulated genes in mouse embryonic fibroblasts.

TFII-I is a founding member of a family of helix-loop-helix transcription factors involved in modulation of genes through interaction with various nuclear factors and chromatin remodeling complexes. Recent studies indicate that TFII-I performs important function in cell physiology and mouse embryogenesis. In order to understand its molecular role, TFII-I was overexpressed in primary mouse embryonic fibroblasts (MEFs) and alterations in gene expression were monitored with a mouse 16 K oligonucleotide microarray. These studies allowed us to identify genes that lie downstream of TFII-I-dependent pathways. Among the modulated candidates were genes involved in the immunity response, catalytic activity, signaling pathways and transcriptional regulation. Expression of several candidates including those for the interferon-stimulated protein (G1p2), small inducible cytokine A7 (Ccl7), ubiquitin-conjugating enzyme 8 (Ube2l6), cysteine-rich protein (Csrp2) and Drosophila delta-like 1 homolog (Dlk1) were confirmed by real-time PCR. The obtained results suggest that TFII-I participates in multiple signaling and regulatory pathways in MEFs.