Rapid generation of mouse model for emerging infectious disease with the case of severe COVID-19.

Since the pandemic of COVID-19 has intensely struck human society, small animal model for this infectious disease is in urgent need for basic and pharmaceutical research. Although several COVID-19 animal models have been identified, many of them show either minimal or inadequate pathophysiology after SARS-CoV-2 challenge. Here, we describe a new and versatile strategy to rapidly establish a mouse model for emerging infectious diseases in one month by multi-route, multi-serotype transduction with recombinant adeno-associated virus (AAV) vectors expressing viral receptor. In this study, the proposed approach enables profound and enduring systemic expression of SARS-CoV-2-receptor hACE2 in wild-type mice and renders them vulnerable to SARS-CoV-2 infection. Upon virus challenge, generated AAV/hACE2 mice showed pathophysiology closely mimicking the patients with severe COVID-19. The efficacy of a novel therapeutic antibody cocktail RBD-chAbs for COVID-19 was tested and confirmed by using this AAV/hACE2 mouse model, further demonstrating its successful application in drug development.

Upregulation of PD-L1 by SARS-CoV-2 promotes immune evasion.

Patients with severe COVID-19 often suffer from lymphopenia, which is linked to T-cell sequestration, cytokine storm, and mortality. However, it remains largely unknown how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces lymphopenia. Here, we studied the transcriptomic profile and epigenomic alterations involved in cytokine production by SARS-CoV-2-infected cells. We adopted a reverse time-order gene coexpression network approach to analyze time-series RNA-sequencing data, revealing epigenetic modifications at the late stage of viral egress. Furthermore, we identified SARS-CoV-2-activated nuclear factor-κB (NF-κB) and interferon regulatory factor 1 (IRF1) pathways contributing to viral infection and COVID-19 severity through epigenetic analysis of H3K4me3 chromatin immunoprecipitation sequencing. Cross-referencing our transcriptomic and epigenomic data sets revealed that coupling NF-κB and IRF1 pathways mediate programmed death ligand-1 (PD-L1) immunosuppressive programs. Interestingly, we observed higher PD-L1 expression in Omicron-infected cells than SARS-CoV-2 infected cells. Blocking PD-L1 at an early stage of virally-infected AAV-hACE2 mice significantly recovered lymphocyte counts and lowered inflammatory cytokine levels. Our findings indicate that targeting the SARS-CoV-2-mediated NF-κB and IRF1-PD-L1 axis may represent an alternative strategy to reduce COVID-19 severity.

A booster dose of Delta × Omicron hybrid mRNA vaccine produced broadly neutralizing antibody against Omicron and other SARS-CoV-2 variants.

BACKGROUND: With the continuous emergence of new SARS-CoV-2 variants that feature increased transmission and immune escape, there is an urgent demand for a better vaccine design that will provide broader neutralizing efficacy. METHODS: We report an mRNA-based vaccine using an engineered "hybrid" receptor binding domain (RBD) that contains all 16 point-mutations shown in the currently prevailing Omicron and Delta variants. RESULTS: A booster dose of hybrid vaccine in mice previously immunized with wild-type RBD vaccine induced high titers of broadly neutralizing antibodies against all tested SARS-CoV-2 variants of concern (VOCs). In naïve mice, hybrid vaccine generated strong Omicron-specific neutralizing antibodies as well as low but significant titers against other VOCs. Hybrid vaccine also elicited CD8+/IFN-γ+ T cell responses against a conserved T cell epitope present in wild type and all VOCs. CONCLUSIONS: These results demonstrate that inclusion of different antigenic mutations from various SARS-CoV-2 variants is a feasible approach to develop cross-protective vaccines.

Gal-1 (Galectin-1) Upregulation Contributes to Abdominal Aortic Aneurysm Progression by Enhancing Vascular Inflammation.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is a vascular degenerative disease causing sudden rupture of aorta and significant mortality in elders. Nevertheless, no prognostic and therapeutic target is available for disease management. Gal-1 (galectin-1) is a ß-galactoside-binding lectin constitutively expressed in vasculature with roles in maintaining vascular homeostasis. This study aims to investigate the potential involvement of Gal-1 in AAA progression. Approach and Results: Gal-1 was significantly elevated in circulation and aortic tissues of Ang II (angiotensin II)-infused apoE-deficient mice developing AAA. Gal-1 deficiency reduced incidence and severity of AAA with lower expression of aortic MMPs (matrix metalloproteases) and proinflammatory cytokines. TNFα (tumor necrosis factor alpha) induced Gal-1 expression in cultured vascular smooth muscle cells and adventitial fibroblasts. Gal-1 deletion enhanced TNFα-induced MMP9 expression in fibroblasts but not vascular smooth muscle cells. Cysteinyl-labeling assay demonstrated that aortic Gal-1 exhibited susceptibility to oxidation in vivo. Recombinant oxidized Gal-1 induced expression of MMP9 and inflammatory cytokines to various extents in macrophages, vascular smooth muscle cells, and fibroblasts through activation of MAP (mitogen-activated protein) kinase signaling. Clinically, serum MMP9 level was significantly higher in both patients with AAA and coronary artery disease than in control subjects, whereas serum Gal-1 level was elevated in patients with AAA but not coronary artery disease when compared with controls. CONCLUSIONS: Gal-1 is highly induced and contributes to AAA by enhancing matrix degradation activity and inflammatory responses in experimental model. The pathological link between Gal-1 and AAA is also observed in human patients. These findings support the potential of Gal-1 as a disease biomarker and therapeutic target of AAA.

Siglec-E retards atherosclerosis by inhibiting CD36-mediated foam cell formation.

BACKGROUND: The accumulation of lipid-laden macrophages, foam cells, within sub-endothelial intima is a key feature of early atherosclerosis. Siglec-E, a mouse orthologue of human Siglec-9, is a sialic acid binding lectin predominantly expressed on the surface of myeloid cells to transduce inhibitory signal via recruitment of SH2-domain containing protein tyrosine phosphatase SHP-1/2 upon binding to its sialoglycan ligands. Whether Siglec-E expression on macrophages impacts foam cell formation and atherosclerosis remains to be established. METHODS: ApoE-deficient (apoE-/-) and apoE/Siglec-E-double deficient (apoE-/-/Siglec-E-/-) mice were placed on high fat diet for 3 months and their lipid profiles and severities of atherosclerosis were assessed. Modified low-density lipoprotein (LDL) uptake and foam cell formation in wild type (WT) and Siglec-E-/-- peritoneal macrophages were examined in vitro. Potential Siglec-E-interacting proteins were identified by proximity labeling in conjunction with proteomic analysis and confirmed by coimmunoprecipitation experiment. Impacts of Siglec-E expression and cell surface sialic acid status on oxidized LDL uptake and signaling involved were examined by biochemical assays. RESULTS: Here we show that genetic deletion of Siglec-E accelerated atherosclerosis without affecting lipid profile in apoE-/- mice. Siglec-E deficiency promotes foam cell formation by enhancing acetylated and oxidized LDL uptake without affecting cholesterol efflux in macrophages in vitro. By performing proximity labeling and proteomic analysis, we identified scavenger receptor CD36 as a cell surface protein interacting with Siglec-E. Further experiments performed in HEK293T cells transiently overexpressing Siglec-E and CD36 and peritoneal macrophages demonstrated that depletion of cell surface sialic acids by treatment with sialyltransferase inhibitor or sialidase did not affect interaction between Siglec-E and CD36 but retarded Siglec-E-mediated inhibition on oxidized LDL uptake. Subsequent experiments revealed that oxidized LDL induced transient Siglec-E tyrosine phosphorylation and recruitment of SHP-1 phosphatase in macrophages. VAV, a downstream effector implicated in CD36-mediated oxidized LDL uptake, was shown to interact with SHP-1 following oxidized LDL treatment. Moreover, oxidized LDL-induced VAV phosphorylation was substantially lower in WT macrophages comparing to Siglec-E-/- counterparts. CONCLUSIONS: These data support the protective role of Siglec-E in atherosclerosis. Mechanistically, Siglec-E interacts with CD36 to suppress downstream VAV signaling involved in modified LDL uptake.

TRC8 downregulation contributes to the development of non-alcoholic steatohepatitis by exacerbating hepatic endoplasmic reticulum stress.

Endoplasmic reticulum (ER) stress is implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). TRC8 is an ER-resident E3 ligase with roles in modulating lipid and protein biosynthesis. In this study we showed that TRC8 expression was downregulated in steatotic livers of patients and mice fed with a high fat diet (HFD) or a methionine and choline deficient (MCD) diet. To investigate the impact of TRC8 downregulation on steatosis and the progression to non-alcoholic steatohepatitis (NASH), we placed TRC8 knockout (KO) mice and wild type (WT) controls on a HFD or MCD diet and the severities of steatosis and NASH developed were compared. We found that TRC8 deficiency did not significantly affect diet-induced steatosis. Nevertheless, MCD diet-induced NASH as characterized by hepatocyte death, inflammation and fibrosis were exacerbated in TRC8-KO mice. The hepatic ER stress response, as evidenced by increased eIF2α phosphorylation and expression of ATF4 and CHOP, and the level of activated caspase 3, an apoptosis indicator, were augmented by TRC8 deficiency. The hepatic ER stress and NASH induced in mice could be ameliorated by adenovirus-mediated hepatic TRC8 overexpression. Mechanistically, we found that TRC8 deficiency augmented lipotoxic-stress-induced unfolded protein response in hepatocytes by attenuating the arrest of protein translation and the misfolded protein degradation. These findings disclose a crucial role of TRC8 in the maintenance of ER protein homeostasis and its downregulation in steatotic liver contributes to the progression of NAFLD.

Myeloid heme oxygenase-1 promotes metastatic tumor colonization in mice.

Heme oxygenase-1 (HO-1) is a heme degradation enzyme with antioxidant and immune-modulatory functions. HO-1 promotes tumorigenesis by enhancing tumor cell proliferation and invasion. Whether HO-1 has an effect on cancer progression through stromal compartments is less clear. Here we show that the growth of tumor engrafted subcutaneously in syngeneic mice was not affected by host HO-1 expression. However, lung metastasis arisen from subcutaneous tumor or circulating tumor cells was significantly reduced in HO-1(+/-) mice comparing to wild type (WT) mice. The reduced lung metastasis was also observed in B6 mice bearing HO-1(+/-) bone marrow as comparing to WT chimeras, indicating that HO-1 expression in hematopoietic cells impacts tumor colonization at the metastatic site. Further experiments demonstrated that the numbers of myeloid cells recruited to pulmonary premetastatic niches and metastatic loci were significantly lower in HO-1(+/-) mice than in WT mice. Likewise, the extents of tumor cell extravasation and colonization at the metastatic loci in the early phase of metastasis were significantly lower in HO-1(+/-) mice. Mechanistic studies revealed that HO-1 impacted chemoattractant-induced myeloid cell migration by modulating p38 kinase signaling. Moreover, myeloid HO-1-induced expressions of vascular endothelial growth factor and interleukin-10 promoted tumor cell transendothelial migration and STAT3 activation in vitro. These data support a pathological role of myeloid HO-1 in metastasis and suggest a possibility of targeting myeloid HO-1 for cancer treatment.

Activator protein-2α mediates carbon monoxide-induced stromal cell-derived factor-1α expression and vascularization in ischemic heart.

OBJECTIVE: Increased cardiac stromal cell-derived factor-1α (SDF-1α) expression promotes neovascularization and myocardial repair after ischemic injury through recruiting stem cells and reducing cardiomyocyte death. Previous studies have shown that heme oxygenase-1 and its reaction byproduct, carbon monoxide (CO), induce SDF-1α expression in ischemic heart. However, the mechanism underlying heme oxygenase-1/CO-induced cardiac SDF-1α expression remains elusive. This study aims to investigate the signaling pathway and the transcriptional factor that mediate CO-induced SDF-1α gene expression and cardioprotection. APPROACH AND RESULTS: CO gas and a CO-releasing compound, tricarbonyldichlororuthenium (II) dimer, dose-dependently induced SDF-1α expression in primary neonatal cardiomyocytes and H9C2 cardiomyoblasts. Promoter luciferase-reporter assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation demonstrated that the activator protein 2α (AP-2α) mediated tricarbonyldichlororuthenium (II) dimer-induced SDF-1α gene transcription. Tricarbonyldichlororuthenium (II) dimer induced AP-2α expression via protein kinase B (AKT)-dependent signaling. AKT inhibition or AP-2α knockdown reduced tricarbonyldichlororuthenium (II) dimer-induced SDF-1α expression. Coronary ligation induced transient increases of cardiac AP-2α and SDF-1α expression, which were declined at 1 week postinfarction in mice. Periodic exposure of coronary-ligated mice to CO (250 ppm for 1 hour/day, 6 days) resumed the induction of AP-2α and SDF-1α gene expression in infarcted hearts. Immunohistochemistry and echocardiography performed at 4 weeks after coronary ligation revealed that CO treatment enhanced neovascularization in the myocardium of peri-infarct region and improved cardiac function. CO-mediated SDF-1α expression and cardioprotection was ablated by intramyocardial injection of lentivirus bearing specific short hairpin RNA targeting AP-2α. CONCLUSIONS: Our data demonstrate that AKT-dependent upregulation of AP-2α is essential for CO-induced SDF-1α expression and myocardial repair after ischemic injury.

Optimization and Improvement of qPCR Detection Sensitivity of SARS-CoV-2 in Saliva.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been a major public health threat globally, especially during the beginning of the pandemic in 2020. Reverse transcription-quantitative PCR (RT-qPCR) is utilized for viral RNA detection as part of control measures to limit the spread of COVID-19. Collecting nasopharyngeal swabs for RT-qPCR is a routine diagnostic method for COVID-19 in clinical settings, but its large-scale implementation is hindered by a shortage of trained health professionals. Despite concerns over its sensitivity, saliva has been suggested as a practical alternative sampling approach to the nasopharyngeal swab for viral RNA detection. In this study, we spiked saliva from healthy donors with inactivated SARS-CoV-2 from an international standard to evaluate the effect of saliva on viral RNA detection. On average, the saliva increased the cycle threshold (CT) values of the SARS-CoV-2 RNA samples by 2.64 compared to the viral RNA in viral transport medium. Despite substantial variation among different donors in the effect of saliva on RNA quantification, the outcome of the RT-qPCR diagnosis was largely unaffected for viral RNA samples with CT values of <35 (1.55 log10 IU/mL). The saliva-treated viral RNA remained stable for up to 6 h at room temperature and 24 h at 4°C. Further supplementing protease and RNase inhibitors improved the detection of viral RNA in the saliva samples. Our data provide practical information on the storage conditions of saliva samples and suggest optimized sampling procedures for SARS-CoV-2 diagnosis. IMPORTANCE The primary method for detection of SARS-CoV-2 is using nasopharyngeal swabs, but a shortage of trained health professionals has hindered its large-scale implementation. Saliva-based nucleic acid detection is a widely adopted alternative, due to its convenience and minimally invasive nature, but the detection limit and direct impact of saliva on viral RNA remain poorly understood. To address this gap in knowledge, we used a WHO international standard to evaluate the effect of saliva on SARS-CoV-2 RNA detection. We describe the detection profile of saliva-treated SARS-CoV-2 samples under different storage temperatures and incubation periods. We also found that adding protease and RNase inhibitors could improve viral RNA detection in saliva. Our research provides practical recommendations for the optimal storage conditions and sampling procedures for saliva-based testing, which can improve the efficiency of COVID-19 testing and enhance public health responses to the pandemic.

Ubiquitin-proteasome system mediates heme oxygenase-1 degradation through endoplasmic reticulum-associated degradation pathway.

The present study investigated the cellular mechanism underlying the degradation of heme oxygenase-1 (HO-1), an endoplasmic reticulum (ER)-anchored protein. The turnover of HO-1 induced in vascular smooth muscle cells (VSMCs) was significantly attenuated by proteasome inhibitors, suggesting the involvement of a proteasome-mediated pathway. High molecular weight ubiquitin conjugates were co-immunoprecipitated with HO-1 from VSMCs after proteasome inhibition, and HO-1 ubiquitination was confirmed in HEK293 cells overexpressing His-tagged HO-1 and HA-tagged ubiquitin. Endogenous p97, an ATPase, and Ufd1, both implicated as essential components in the ER-associated degradation pathway (ERAD), were co-eluted with His-tagged HO-1 from metal affinity resin. Knockdown of either p97 or Ufd1 in HEK293 cells using specific siRNA significantly prolonged the half-life of endogenously induced HO-1 and slowed the degradation of ubiquitinated HO-1. HO-1 ubiquitination in HEK293 cells was enhanced by zinc chloride, but suppressed with a zinc chelator (N,N,N',N'-tetrakis(2-pyridylmethyl)ethyl-enediamine), suggesting the involvement of a RING-E3 ligase in this process. Collectively, these data indicate that HO-1 protein turnover is regulated by the ubiquitin-proteasome system through the ERAD pathway.

Signal peptide peptidase promotes tumor progression via facilitating FKBP8 degradation.

Signal peptide peptidase (SPP) is an endoplasmic reticulum (ER)-resident aspartyl protease mediating intramembrane cleavage of type II transmembrane proteins. Increasing evidence has supported the role of SPP in ER-associated protein degradation. In the present study, we show that SPP expression is highly induced in human lung and breast cancers and correlated with disease outcome. Stable depletion of SPP expression in lung and breast cancer cell lines significantly reduced cell growth and migration/invasion abilities. Quantitative analysis of the proteomic changes of microsomal proteins in lung cancer cells by the stable isotope labeling with amino acids in cell culture (SILAC) approach revealed that the level of FKBP8, an endogenous inhibitor of mTOR, was significantly increased following SPP depletion. Co-immunoprecipitation assay and confocal immunofluorescence demonstrated that SPP interacted and colocalized with FKBP8 in ER, supporting that FKBP8 is a protein substrate of SPP. Cycloheximide chase and proteasome inhibition experiments revealed that SPP-mediated proteolysis facilitated FKBP8 protein degradation in cytosol. Further experiment demonstrated that the levels of phosphorylation in mTOR and its downstream effectors, S6K and 4E-BP1, were significantly lower in SPP-depleted cells. The reduced mTOR signaling and decreases of growth and migration/invasion abilities induced by SPP depletion in cancer cells could be reversed by FKBP8 downregulation. The implication of FKBP8 in SPP-mediated tumorigenicity was also observed in the xenograft model. Together, these findings disclose that SPP promotes tumor progression, at least in part, via facilitating the degradation of FKBP8 to enhance mTOR signaling.

Galectin-1 Restricts Vascular Smooth Muscle Cell Motility Via Modulating Adhesion Force and Focal Adhesion Dynamics.

Vascular smooth muscle cell (VSMC) migration play a key role in the development of intimal hyperplasia and atherosclerosis. Galectin-1 (Gal-1) is a redox-sensitive ß-galactoside-binding lectin expressed in VSMCs with intracellular and extracellular localizations. Here we show that VSMCs deficient in Gal-1 (Gal-1-KO) exhibited greater motility than wild type (WT) cells. Likewise, Gal-1-KO-VSMC migration was inhibited by a redox-insensitive but activity-preserved Gal-1 (CSGal-1) in a glycan-dependent manner. Gal-1-KO-VSMCs adhered slower than WT cells on fibronectin. Cell spreading and focal adhesion (FA) formation examined by phalloidin and vinculin staining were less in Gal-1-KO-VSMCs. Concomitantly, FA kinase (FAK) phosphorylation was induced to a lower extent in Gal-1-KO cells. Analysis of FA dynamics by nocodazole washout assay demonstrated that FA disassembly, correlated with FAK de-phosphorylation, was faster in Gal-1-KO-VSMCs. Surface plasmon resonance assay demonstrated that CSGal-1 interacted with α5ß1integrin and fibronectin in a glycan-dependent manner. Chemical crosslinking experiment and atomic force microscopy further revealed the involvement of extracellular Gal-1 in strengthening VSMC-fibronectin interaction. In vivo experiment showed that carotid ligation-induced neointimal hyperplasia was more severe in Gal-1-KO mice than WT counterparts. Collectively, these data disclose that Gal-1 restricts VSMC migration by modulating cell-matrix interaction and focal adhesion turnover, which limits neointimal formation post vascular injury.

Heme oxygenase-1 inhibits angiotensin II-induced cardiac hypertrophy in vitro and in vivo.

BACKGROUND: Heme oxygenase-1 (HO-1) is a stress-response enzyme implicated in cardioprotection. To explore whether HO-1 has a role in cardiac remodeling response, the effect of its overexpression on angiotensin II (Ang II)-induced cardiac hypertrophy was examined. METHODS AND RESULTS: HO-1 was induced in cultured rat neonatal cardiomyocytes by treatment with cobalt protoporphyrin IX (CoPPIX) or a recombinant adenovirus carrying the human HO-1 gene. Ang II-induced myocyte hypertrophy assessed by increments in cell size, [3H]leucine uptake, and protein content was suppressed by HO-1 overexpression. Cotreatment of cells with tin protoporphyrin IX, a HO inhibitor, significantly reversed the suppressive effect of HO-1. Bilirubin, one of the byproducts of heme degradation by HO-1, mediated the suppressive effect through the inhibition of Ang II-induced production of reactive oxygen species, as detected by a 2',7'-dichlorofluorescein probe. The antihypertrophic effect of HO-1 was also demonstrated in rats receiving chronic Ang II infusions. Cotreatment of animals with CoPPIX significantly attenuated Ang II-induced left ventricular hypertrophy and hyperdynamic contractions, whereas concomitant treatment with tin protoporphyrin IX abolished CoPPIX-mediated cardioprotection in vivo. CONCLUSIONS: HO-1 attenuates Ang II-induced cardiac hypertrophy both in vitro and in vivo, and bilirubin mediates, at least in part, the antihypertrophic effect of HO-1 via inhibition of reactive oxygen species production after Ang II stimulation.

Adipose overexpression of heme oxygenase-1 does not protect against high fat diet-induced insulin resistance in mice.

Heme oxygenase-1 (HO-1) is a stress-responsive enzyme with potent anti-oxidant and anti-inflammatory activities. Previous studies have shown that systemic induction of HO-1 by chemical inducers reduces adiposity and improves insulin sensitivity. To dissect the specific function of HO-1 in adipose tissue, we generated transgenic mice with adipose HO-1 overexpression using the adipocyte-specific aP2 promoter. The transgenic (Tg) mice exhibit similar metabolic phenotype as wild type (WT) control under chow-fed condition. High fat diet (HFD) challenge significantly increased the body weights of WT and Tg mice to a similar extent. Likewise, HFD-induced glucose intolerance and insulin resistance were not much different between WT and Tg mice. Analysis of the adipose tissue gene expression revealed that the mRNA levels of adiponectin and interleukin-10 were significantly higher in chow diet-fed Tg mice as compared to WT counterparts, whereas HFD induced downregulation of adiponectin gene expression in both Tg and WT mice to a similar level. HFD-induced proinflammatory cytokine expression in adipose tissues were comparable between WT and transgenic mice. Nevertheless, immunohistochemistry and gene expression analysis showed that the number of infiltrating macrophages with preferential expression of M2 markers was significantly higher in the adipose tissue of obese Tg mice than WT mice. Further experiment demonstrated that myeloid cells from Tg mice expressed higher level of HO-1 and exhibited greater migration response toward chemoattractant in vitro. Collectively, these data indicate that HO-1 overexpression in adipocytes does not protect against HFD-induced obesity and the development of insulin resistance in mice.

Myeloid heme oxygenase-1 haploinsufficiency reduces high fat diet-induced insulin resistance by affecting adipose macrophage infiltration in mice.

Increased adipose tissue macrophages contribute to obesity-induced metabolic syndrome. Heme oxygenase-1 (HO-1) is a stress-inducible enzyme with potent anti-inflammatory and proangiogenic activities in macrophages. However, the role of macrophage HO-1 on obesity-induced adipose inflammation and metabolic syndrome remains unclear. Here we show that high-fat diet (HFD) feeding in C57BL/6J mice induced HO-1 expression in the visceral adipose tissue, particularly the stromal vascular fraction. When the irradiated C57BL/6J mice reconstituted with wild-type or HO-1(+/-) bone marrow were fed with HFD for over 24 weeks, the HO-1(+/-) chimeras were protected from HFD-induced insulin resistance and this was associated with reduced adipose macrophage infiltration and angiogenesis, suggesting that HO-1 affects myeloid cell migration toward adipose tissue during obesity. In vivo and in vitro migration assays revealed that HO-1(+/-) macrophages exhibited an impaired migration response. Chemoattractant-induced phosphorylation of p38 and focal adhesion kinase (FAK) declined faster in HO-1(+/-) macrophages. Further experiments demonstrated that carbon monoxide and bilirubin, the byproducts derived from heme degradation by HO-1, enhanced macrophage migration by increasing phosphorylation of p38 and FAK, respectively. These data disclose a novel role of hematopoietic cell HO-1 in promoting adipose macrophage infiltration and the development of insulin resistance during obesity.

Systemic expression of heme oxygenase-1 ameliorates type 1 diabetes in NOD mice.

Heme oxygenase-1 (HO-1) is an enzyme with potent immunoregulatory capacity. To evaluate the effect of HO-1 on autoimmune diabetes, female NOD mice at 9 weeks of age received a single intravenous injection of a recombinant adeno-associated virus bearing HO-1 gene (AAV-HO-1; 0.5 x 10(10)-2.5 x 10(10) viruses/mouse). In a dose-dependent manner, HO-1 transduction reduced destructive insulitis and the incidence of overt diabetes examined over a 15-week period. HO-1-mediated protection was associated with a lower type 1 T-helper cell (Th1)-mediated response. Adaptive transfer experiments in NOD.scid mice demonstrated that splenocytes isolated from AAV-HO-1-treated mice were less diabetogenic. Flow cytometry analysis revealed no significant difference in the percentages of CD4(+)CD25(+) regulatory T-cells between saline-treated and AAV-HO-1-treated groups. However, the CD11c(+) major histocompatibility complex II(+) dendritic cell population was much lower in the AAV-HO-1-treated group. A similar protective effect against diabetes was observed in NOD mice subjected to carbon monoxide (CO) gas (250 ppm CO for 2 h, twice per week). These data suggest that HO-1 slows the progression to overt diabetes in pre-diabetic NOD mice by downregulating the phenotypic maturity of dendritic cells and Th1 effector function. CO appears to mediate at least partly the beneficial effect of HO-1 in this disease setting.

Carbon monoxide-induced early thrombolysis contributes to heme oxygenase-1-mediated inhibition of neointimal growth after vascular injury in hypercholesterolemic mice.

Arterial thrombosis is a critical event in the pathogenesis of lesion development. In this study, we evaluated the effect of heme oxygenase-1 (HO-1), a stress-inducible enzyme with vasoprotective functions, on arterial thrombosis following vascular mechanical injury. The carotid arteries of apoE-deficient mice were subjected to angioplasty with a modified beaded-needle. Arterial thrombosis occurred at 12 h after injury. Treatment of the injured vessels with an adenovirus bearing HO-1 gene (Adv-HO-1) (1 x 10(8) pfu), but not saline or empty adenovirus (Adv), immediately after angioplasty resulted in earlier thrombolysis and restoration of blood flow detected at 24 h. Immunohistochemistry revealed that the arterial plasminogen activator inhibitor-1 (PAI-1) expression was markedly reduced in Adv-HO-1-treated injured arteries as compared to control counterparts. The thrombolytic effect was also observed by exposing animals with existing arterial thrombosis to carbon monoxide (CO) (250 ppm, 2 h), a byproduct derived from heme degradation by HO-1. In parallel with less fibrin(ogen) deposition, the macrophage infiltration, monocyte chemoattractant protein-1 expression and neointimal formation assessed at 2 weeks after angioplasty were substantially reduced in injured arteries treated with Adv-HO-1. These results support a role of early thrombolysis induced by CO in HO-1-mediated protection against intimal hyperplasia after vascular injury.