Adenoviruses are used extensively as gene transfer agents, both experimentally and clinically. However, targeting of liver cells by adenoviruses compromises their potential efficacy. In cell culture, the adenovirus serotype 5 fiber protein engages the coxsackievirus and adenovirus receptor (CAR) to bind cells. Paradoxically, following intravascular delivery, CAR is not used for liver transduction, implicating alternate pathways. Recently, we demonstrated that coagulation factor (F)X directly binds adenovirus leading to liver infection. Here, we show that FX binds to the Ad5 hexon, not fiber, via an interaction between the FX Gla domain and hypervariable regions of the hexon surface. Binding occurs in multiple human adenovirus serotypes. Liver infection by the FX-Ad5 complex is mediated through a heparin-binding exosite in the FX serine protease domain. This study reveals an unanticipated function for hexon in mediating liver gene transfer in vivo.

Atherosclerosis is a chronic inflammatory disease of the vasculature commonly leading to myocardial infarction and stroke. We show that IL-33, which is a novel IL-1–like cytokine that signals via ST2, can reduce atherosclerosis development in ApoE−/− mice on a high-fat diet. IL-33 and ST2 are present in the normal and atherosclerotic vasculature of mice and humans. Although control PBS-treated mice developed severe and inflamed atherosclerotic plaques in the aortic sinus, lesion development was profoundly reduced in IL-33–treated animals. IL-33 also markedly increased levels of IL-4, -5, and -13, but decreased levels of IFNγ in serum and lymph node cells. IL-33 treatment also elevated levels of total serum IgA, IgE, and IgG1, but decreased IgG2a, which is consistent with a Th1-to-Th2 switch. IL-33–treated mice also produced significantly elevated antioxidized low-density lipoprotein (ox-LDL) antibodies. Conversely, mice treated with soluble ST2, a decoy receptor that neutralizes IL-33, developed significantly larger atherosclerotic plaques in the aortic sinus of the ApoE−/− mice compared with control IgG-treated mice. Furthermore, coadministration of an anti–IL-5 mAb with IL-33 prevented the reduction in plaque size and reduced the amount of ox-LDL antibodies induced by IL-33. In conclusion, IL-33 may play a protective role in the development of atherosclerosis via the induction of IL-5 and ox-LDL antibodies.

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Objective-MicroRNAs (miRNAs) are small noncoding RNAs that have the capacity to control protein production through binding "seed" sequences within a target mRNA. Each miRNA is capable of potentially controlling hundreds of genes. The regulation of miRNAs in the lung during the development of pulmonary arterial hypertension (PAH) is unknown. Methods and Results-We screened lung miRNA profiles in a longitudinal and crossover design during the development of PAH caused by chronic hypoxia or monocrotaline in rats. We identified reduced expression of Dicer, involved in miRNA processing, during the onset of PAH after hypoxia. MiR-22, miR-30, and let-7f were downregulated, whereas miR-322 and miR-451 were upregulated significantly during the development of PAH in both models. Differences were observed between monocrotaline and chronic hypoxia. For example, miR-21 and let-7a were significantly reduced only in monocrotaline-treated rats. MiRNAs that were significantly regulated were validated by quantitative polymerase chain reaction. By using in vitro studies, we demonstrated that hypoxia and growth factors implicated in PAH induced similar changes in miRNA expression. Furthermore, we confirmed miR-21 downregulation in human lung tissue and serum from patients with idiopathic PAH. Conclusion-Defined miRNAs are regulated during the development of PAH in rats. Therefore, miRNAs may contribute to the pathogenesis of PAH and represent a novel opportunity for therapeutic intervention. Key Words: pulmonary hypertension Ⅲ small RNA molecules Ⅲ gene regulation P ulmonary arterial hypertension (PAH) is a complex disorder characterized by the obstructive remodeling of pulmonary arteries, leading to a progressive elevation in pulmonary arterial pressure (PAP) and subsequent right-sided heart failure and death. 1 Familial PAH is associated in 80% of cases with diverse heterozygous mutations in the gene-encoding bone morphogenetic protein receptor 2 (BMPR-II) 2 and can be associated with mutations in the activin-receptor kinaselike 1 gene. 3 The cause of the variable phenotypic expression of PAH among carriers of mutated BMPR-II genes is unclear, and is likely related to environmental and genetic modifiers. Although BMPR-II-related pathways are considered pivotal, many other mediator pathways participate in the pathogenesis of PAH and are being actively investigated, both independently and in combination. For example, the involvement of serotonin in the development of experimental PAH has been recently reported. 4,5 Indeed, important interactions between the serotonin and BMP pathways have recently been described. 6 Rats exposed to hypoxia or injected with the toxin monocrotaline develop pulmonary arterial changes correlated with the development of PAH, including remodeling and elevating PAP.MicroRNAs (miRNAs) are small noncoding transcripts of 16 to 29 nucleotide RNAs that regulate gene expression posttranscriptionally by targeting mRNAs. Animal miRNAs are processed from longer primary transcripts (primary miRNAs) that can contain ...

A major limitation for adenoviral transduction in vivo is the profound liver tropism of adenovirus type 5 (Ad5). Recently, we demonstrated that coagulation factor X (FX) binds to Ad5-hexon protein at high affinity to mediate hepatocyte transduction after intravascular delivery. We developed novel genetically FX-binding ablated Ad5 vectors with lower liver transduction. Here, we demonstrate that FX-binding ablated Ad5 predominantly localize to the liver and spleen 1 hour after injection; however, they had highly reduced liver transduction in both control and macrophagedepleted mice compared with Ad5. At high doses in macrophage-depleted mice, FX-binding ablated vectors transduced the spleen more efficiently than Ad5. Immunohistochemical studies demonstrated transgene colocalization with CD11c ؉ , ER-TR7 ؉ , and MAdCAM-1 ؉ cells in the splenic marginal zone. Systemic inflammatory profiles were broadly similar between FX-binding ablated Ad5 and Ad5 at low and intermediate doses, although higher levels of several inflammatory proteins were observed at the highest dose of FX-binding ablated Ad5. Subsequently, we generated a FX-binding ablated virus containing a high affinity Ad35 fiber that mediated a significant improvement in lung/liver ratio in macrophage-depleted CD46 ؉ mice compared with controls. Therefore, this study documents the biodistribution and reports the retargeting capacity of IntroductionOf the 54 different adenoviral serotypes isolated to date, adenovirus serotype 5 (Ad5) has been the most commonly used vector in gene therapy clinical trials. This is, in part, due to clear advantages over alternate strategies including the relatively easy manipulation of its viral genome and feasible scale-up production to high titers (up to 10 13 viral particles (vp)/mL). Nevertheless, Ad5 presents 2 substantial limitations that have required attention to optimize the use of Ad5 in gene therapy. These include the observation that the majority of the human population has pre-existing neutralizing antibodies against Ad5 1-3 and the profound liver tropism observed for Ad5 after intravascular delivery. 4,5 For this reason, fundamental aspects of Ad5 biology need to be further studied to provide safer and target-specific Ad5 gene therapy vectors. The mechanism of Ad5-mediated gene transfer has now been relatively well characterized. In vitro studies have shown that Ad5 and those Ads from subspecies A, C, D, E, and F may use the coxsackievirus and Ad receptor (CAR) as a primary binding receptor. [6][7][8][9] This interaction occurs via the fiber knob domain with subsequent interaction of the Ad5 penton base with cellular integrins (␣v␤3 and ␣v␤5), mediating capsid internalization. 10,11 Although CAR and integrin-binding ablated mutant Ad vectors show a substantial reduction in transduction in vitro, these vectors still predominantly transduce hepatocytes in vivo after intravascular administration. 12,13 Injection of Ad5 into the bloodstream leads to a complex series of interactions that impact on the resulting biodistri...

BackgroundLittle is known about the roles of myeloid cell subsets in kidney injury and in the limited ability of the organ to repair itself. Characterizing these cells based only on surface markers using flow cytometry might not provide a full phenotypic picture. Defining these cells at the single-cell, transcriptomic level could reveal myeloid heterogeneity in the progression and regression of kidney disease.MethodsIntegrated droplet– and plate-based single-cell RNA sequencing were used in the murine, reversible, unilateral ureteric obstruction model to dissect the transcriptomic landscape at the single-cell level during renal injury and the resolution of fibrosis. Paired blood exchange tracked the fate of monocytes recruited to the injured kidney.ResultsA single-cell atlas of the kidney generated using transcriptomics revealed marked changes in the proportion and gene expression of renal cell types during injury and repair. Conventional flow cytometry markers would not have identified the 12 myeloid cell subsets. Monocytes recruited to the kidney early after injury rapidly adopt a proinflammatory, profibrotic phenotype that expresses Arg1, before transitioning to become Ccr2+ macrophages that accumulate in late injury. Conversely, a novel Mmp12+ macrophage subset acts during repair.ConclusionsComplementary technologies identified novel myeloid subtypes, based on transcriptomics in single cells, that represent therapeutic targets to inhibit progression or promote regression of kidney disease.

Transforming growth factor (TGF)-

Renal tubulointerstitial fibrosis is the common end point of progressive renal disease. MicroRNA (miR)-214 and miR-21 are upregulated in models of renal injury, but the function of miR-214 in this setting and the effect of its manipulation remain unknown. We assessed the effect of inhibiting miR-214 in an animal model of renal fibrosis. In mice, genetic deletion of miR-214 significantly attenuated interstitial fibrosis induced by unilateral ureteral obstruction (UUO). Treatment of wild-type mice with an anti-miR directed against miR-214 (anti-miR-214) before UUO resulted in similar antifibrotic effects, and in vivo biodistribution studies demonstrated that anti-miR-214 accumulated at the highest levels in the kidney. Notably, in vivo inhibition of canonical TGF-b signaling did not alter the regulation of endogenous miR-214 or miR-21. Whereas miR-21 antagonism blocked Smad 2/3 activation, miR-214 antagonism did not, suggesting that miR-214 induces antifibrotic effects independent of Smad 2/3. Furthermore, TGF-b blockade combined with miR-214 deletion afforded additional renal protection. These phenotypic effects of miR-214 depletion were mediated through broad regulation of the transcriptional response to injury, as evidenced by microarray analysis. In human kidney tissue, miR-214 was detected in cells of the glomerulus and tubules as well as in infiltrating immune cells in diseased tissue. These studies demonstrate that miR-214 functions to promote fibrosis in renal injury independent of TGF-b signaling in vivo and that antagonism of miR-214 may represent a novel antifibrotic treatment in the kidney.