Vivo Activation Research Articles

In Vivo Activation of Peritoneal Macrophages in Response to Antibodies in a Murine Model

In Vivo Activation of Peritoneal Macrophages in Response to Antibodies in a Murine Model

In Vivo Activation of Peritoneal Macrophages in Response to Antibodies in a Murine Model

In Vivo Activation of Peritoneal Macrophages in Response to Antibodies in a Murine Model

Direct In Vivo Activation of T Cells with Nanosized Immunofilaments Inhibits Tumor Growth and Metastasis.

Adoptive T cell therapy has successfully been implemented for the treatment of cancer. Nevertheless, ex vivo expansion of T cells by artificial antigen-presenting cells (aAPCs) remains cumbersome and can compromise T cell functionality, thereby limiting their therapeutic potential. We propose a radically different approach aimed at direct expansion of T cells in vivo, thereby omitting the need for large-scale ex vivo T cell production. We engineered nanosized immunofilaments (IFs), with a soluble semiflexible polyisocyanopeptide backbone that presents peptide-loaded major histocompatibility complexes and costimulatory molecules multivalently. IFs readily activated and expanded antigen-specific T cells like natural APCs, as evidenced by transcriptomic analyses of T cells. Upon intravenous injection, IFs reach the spleen and lymph nodes and induce antigen-specific T cell responses in vivo. Moreover, IFs display strong antitumor efficacy resulting in inhibition of the formation of melanoma metastases and reduction of primary tumor growth in synergy with immune checkpoint blockade. In conclusion, nanosized IFs represent a powerful modular platform for direct activation and expansion of antigen-specific T cells in vivo, which can greatly contribute to cancer immunotherapy.

Leptin Elicits In Vivo Eosinophil Migration and Activation: Key Role of Mast Cell-Derived PGD2.

Eosinophils are key regulators of adipose tissue homeostasis, thus characterization of adipose tissue-related molecular factors capable of regulating eosinophil activity is of great interest. Leptin is known to directly activate eosinophils in vitro, but leptin ability of inducing in vivo eosinophilic inflammatory response remains elusive. Here, we show that leptin elicits eosinophil influx as well as its activation, characterized by increased lipid body biogenesis and LTC4 synthesis. Such leptin-triggered eosinophilic inflammatory response was shown to be dependent on activation of the mTOR signaling pathway, since it was (i) inhibited by rapamycin pre-treatment and (ii) reduced in PI3K-deficient mice. Local infiltration of activated eosinophils within leptin-driven inflammatory site was preceded by increased levels of classical mast cell-derived molecules, including TNFα, CCL5 (RANTES), and PGD2. Thus, mice were pre-treated with a mast cell degranulating agent compound 48/80 which was capable to impair leptin-induced PGD2 release, as well as eosinophil recruitment and activation. In agreement with an indirect mast cell-driven phenomenon, eosinophil accumulation induced by leptin was abolished in TNFR-1 deficient and also in HQL-79–pretreated mice, but not in mice pretreated with neutralizing antibodies against CCL5, indicating that both typical mast cell-driven signals TNFα and PGD2, but not CCL5, contribute to leptin-induced eosinophil influx. Distinctly, leptin-induced eosinophil lipid body (lipid droplet) assembly and LTC4 synthesis appears to depend on both PGD2 and CCL5, since both HQL-79 and anti-CCL5 treatments were able to inhibit these eosinophil activation markers. Altogether, our data show that leptin triggers eosinophilic inflammation in vivo via an indirect mechanism dependent on activation of resident mast cell secretory activity and mediation by TNFα, CCL5, and specially PGD2.

In Vivo Activation of Pro-Protein Therapeutics via Chemically Engineered Enzyme Cascade Reaction

Selective and temporal control over protein activity is of great importance for the advancement of the protein of interest into precise molecular medicine. Simply installing synthetic ligands to pr...

In Vivo Activation and Pro-Fibrotic Function of NF-κB in Fibroblastic Cells During Pulmonary Inflammation and Fibrosis Induced by Carbon Nanotubes.

Exposure to insoluble particles in the lung elicits inflammatory responses that eliminate deposited particulates and repair damaged tissue. Overzealous or prolonged responses lead to chronic conditions, such as fibrosis and malignancy, which are frequently progressive and refractory to drug therapy leading to high rates of disability and mortality. The molecular events underlying the progression of lung inflammation to chronic pathology, in particular, the conversion to fibrosis, remain poorly understood. Fibrogenic multi-walled carbon nanotubes (MWCNTs) have been shown to stimulate prominent acute inflammation that evolves into chronic lesions characterized by chronic inflammation, interstitial fibrosis, and granulomas in mouse lungs. In this communication, we examined the in vivo activation of nuclear factor-κB (NF-κB) signaling in fibroblastic cells during the inflammatory and fibrotic progression induced by MWCNTs. Wild-type C57BL/6J male mice were exposed to two fibrogenic MWCNTs (Mitsui XNRI MWNT-7 and long MWCNTs) by pharyngeal aspiration. Both MWCNTs strongly stimulated the nuclear translocation of NF-κB p65 in lung fibroblasts and myofibroblasts during the acute and chronic responses. Phosphorylated NF-κB p65 at serine 276, a marker of NF-κB activation, was markedly induced by MWCNTs in the nucleus of fibroblastic cells. Moreover, two NF-κB-regulated genes encoding pro-fibrotic mediators, tissue inhibitor of metalloproteinase 1 (TIMP1), and osteopontin (OPN), respectively, were significantly induced in lung fibroblasts and myofibroblasts. These results demonstrate that NF-κB is activated to mediate transactivation of pro-fibrotic genes in fibroblastic cells during pulmonary acute and chronic responses to CNTs, providing a mechanistic framework for analyzing gene regulation in pulmonary fibrotic progression through NF-κB signaling.

Lysophosphatidic Acid Enhances In Vivo Infiltration and Activation of Guinea Pig Eosinophils and Neutrophils via a Rho/Rho-Associated Protein Kinase-Mediated Pathway

Lysophosphatidic acid (LPA) has been shown to be a chemoattractant in in vitro studies. The present study was carried out to determine whether LPA enhances infiltration of inflammatory cells in in vivo studies with guinea pigs. LPA (1 – 10 μg/ml), when by guinea pigs for 5 min, substantially increased the numbers of eosinophils and neutrophils in the bronchoalveolar lavege fluid (BALF), which was recovered at over 4 h after the inhalation of LPA. Infiltration in BALF was significantly inhibited by inhalation of Y-27632, an inhibitor of Rho-associated protein kinase (ROCK). LPA also increased superoxide production of eosinophils and neutrophils. In contrast, Y-27632 inhibited superoxide production. These findings suggest that LPA may contribute to infiltration and activation of inflammatory cells in bronchial asthma; furthermore, the Rho/ROCK-mediated pathway may be involved.

In Vivo Delivery and Activation of Masked Fluorogenic Hydrolase Substrates by Endogenous Hydrolases in C. elegans.

Protein expression and localization are often studied in vivo by tagging molecules with green fluorescent protein (GFP), yet subtle changes in protein levels are not easily detected. To develop a sensitive in vivo method to amplify fluorescence signals and allow cell-specific quantification of protein abundance changes, we sought to apply an enzyme-activated cellular fluorescence system in vivo by delivering ester-masked fluorophores to Caenorhabditis elegans neurons expressing porcine liver esterase (PLE). To aid uptake into sensory neuron membranes, we synthesized two novel fluorogenic hydrolase substrates with long hydrocarbon tails. Recombinant PLE activated these fluorophores in vitro. In vivo activation occurred in sensory neurons, along with potent activation in intestinal lysosomes quantifiable by imaging and microplate and partially attributable to gut esterase 1 (GES-1) activity. These data demonstrate the promise of biorthogonal hydrolases and their fluorogenic substrates as in vivo neuronal imaging tools and for characterizing endogenous C. elegans hydrolase substrate specificities.

In Vivo Activation of Duocarmycin-Antibody Conjugates by Near-Infrared Light.

Near-IR photocaging groups based on the heptamethine cyanine scaffold present the opportunity to visualize and then treat diseased tissue with potent bioactive molecules. Here we describe fundamental chemical studies that enable biological validation of this approach. Guided by rational design, including computational analysis, we characterize the impact of structural alterations on the cyanine uncaging reaction. A modest change to the ethylenediamine linker (N,N′-dimethyl to N,N′-diethyl) leads to a bathochromic shift in the absorbance maxima, while decreasing background hydrolysis. Building on these structure–function relationship studies, we prepare antibody conjugates that uncage a derivative of duocarmycin, a potent cytotoxic natural product. The optimal conjugate, CyEt-Pan-Duo, undergoes small molecule release with 780 nm light, exhibits activity in the picomolar range, and demonstrates excellent light-to-dark selectivity. Mouse xenograft studies illustrate that the construct can be imaged in vivo prior to uncaging with an external laser source. Significant reduction in tumor burden is observed following a single dose of conjugate and near-IR light. These studies define key chemical principles that enable the identification of cyanine-based photocages with enhanced properties for in vivo drug delivery.

In Vivo Activation of Human NK Cells by Treatment with an Interleukin-15 Superagonist Potently Inhibits Acute In Vivo HIV-1 Infection in Humanized Mice.

Natural killer (NK) cells with anti-HIV-1 activity may inhibit HIV-1 replication and dissemination during acute HIV-1 infection. We hypothesized that the capacity of NK cells to suppress acute in vivo HIV-1 infection would be augmented by activating them via treatment with an interleukin-15 (IL-15) superagonist, IL-15 bound to soluble IL-15Rα, an approach that potentiates human NK cell-mediated killing of tumor cells. In vitro stimulation of human NK cells with a recombinant IL-15 superagonist significantly induced their expression of the cytotoxic effector molecules granzyme B and perforin; their degranulation upon exposure to K562 cells, as indicated by cell surface expression of CD107a; and their capacity to lyse K562 cells and HIV-1-infected T cells. The impact of IL-15 superagonist-induced activation of human NK cells on acute in vivo HIV-1 infection was investigated by using hu-spl-PBMC-NSG mice, NOD-SCID-IL2rγ(-/-) (NSG) mice intrasplenically injected with human peripheral blood mononuclear cells (PBMCs) which develop productive in vivo infection after intrasplenic inoculation with HIV-1. IL-15 superagonist treatment potently inhibited acute HIV-1 infection in hu-spl-PBMC-NSG mice even when delayed until 3 days after intrasplenic HIV-1 inoculation. Removal of NK cells from human PBMCs prior to intrasplenic injection into NSG mice completely abrogated IL-15 superagonist-mediated suppression of in vivo HIV-1 infection. Thus, the in vivo activation of NK cells, integral mediators of the innate immune response, by treatment with an IL-15 superagonist increases their anti-HIV activity and enables them to potently suppress acute in vivo HIV-1 infection. These results indicate that in vivo activation of NK cells may represent a new immunotherapeutic approach to suppress acute HIV-1 infection. Epidemiological studies have indicated that NK cells contribute to the control of HIV-1 infection, and in vitro studies have demonstrated that NK cells can selectively kill HIV-1-infected cells. We demonstrated that in vivo activation of NK cells by treatment with an IL-15 superagonist that potently stimulates the antitumor activity of NK cells markedly inhibited acute HIV-1 infection in humanized mice, even when activation of NK cells by IL-15 superagonist treatment is delayed until 3 days after HIV-1 inoculation. NK cell depletion from PBMCs prior to their intrasplenic injection abrogated the suppression of in vivo HIV-1 infection observed in humanized mice treated with the IL-15 superagonist, demonstrating that activated human NK cells were mediating IL-15 superagonist-induced inhibition of acute HIV-1 infection. Thus, in vivo immunostimulation of NK cells, a promising therapeutic approach for cancer therapy, may represent a new treatment modality for HIV-1-infected individuals, particularly in the earliest stages of infection.

FRI0169 Elevated LBP Levels and in Vivo Activation of Monocytes as Potential Indicators of Bacterial Translocation in Axial Spondyloarthritis

BackgroundSubclinical gut inflammation is often present in axSpA patients [1, 2]. This may result in enhanced translocation of mucosal antigens leading to activation of monocytes which are very sensitive sensors...

Brivanib Attenuates Hepatic Fibrosis In Vivo and Stellate Cell Activation In Vitro by Inhibition of FGF, VEGF and PDGF Signaling

Background and AimsBrivanib is a selective inhibitor of vascular endothelial growth factor receptor (VEGFR) and fibroblast growth factor receptor (FGFR) tyrosine kinases, which are both involved in mechanisms of liver fibrosis. We hypothesized that inhibition of VEGFR and FGFR by brivanib would inhibit liver fibrosis. We therefore examined the effect of brivanib on liver fibrosis in three mouse models of fibrosis.Methods In vivo, we induced liver fibrosis by bile duct ligation (BDL), chronic carbon tetrachloride (CCl4), and chronic thioacetamide (TAA) administration. Liver fibrosis was examined by immunohistochemistry and Western immunoblotting. In vitro, we used LX-2 human hepatic stellate cells (HSCs) to assess the effect of brivanib on stellate cell proliferation and activation.ResultsAfter in vivo induction with BDL, CCl4, and TAA, mice treated with brivanib showed reduced liver fibrosis and decreased expression of collagen Iα1 and α-smooth muscle actin in the liver. In vitro, brivanib decreased proliferation of HSCs induced by platelet-derived growth factor (PDGF), VEGF, and FGF. Brivanib also decreased stellate cell viability and inhibited PDGFBB-induced phosphorylation of its cognate receptor.ConclusionBrivanib reduces liver fibrosis in three different animal models and decreases human hepatic stellate cell activation. Brivanib may represent a novel therapeutic approach to treatment of liver fibrosis and prevention of liver cancer.

Development of a Tumor‐Specific Photoactivatable Doxorubicin Prodrug

This is a retrospective highlight on the publication by Ibsen and coworkers: Localized In Vivo Activation of a Photoactivatable Doxorubicin Prodrug in Deep Tumor Tissue, which appeared in a preceding issue of Photochem. Photobiol. (2013, 89:698-708). The authors describe the synthesis and properties of a novel doxorubicin (DOX) prodrug, DOX-PCB, which contains a photocleavable linker group. Systemic administration of the prodrug to a tumor-bearing animal followed by LED/fiber optic 365nm light delivery allowed active DOX to be released site specifically in the tumor area. This elegant and timely study provides compelling evidence that photocleavable DOX-PCB can eliminate many of the toxic side effects of DOX that have plagued clinical use of this highly effective antitumor drug for many years.

In Vivo Activation of the p53 Tumor Suppressor Pathway by an Engineered Cyclotide

The overexpression of Hdm2 and HdmX is a common mechanism used by many tumor cells to inactive the p53 tumor suppressor pathway promoting cell survival. Targeting Hdm2 and HdmX has emerged as a validated therapeutic strategy for treating cancers with wild-type p53. Small linear peptides mimicking the N-terminal fragment of p53 have been shown to be potent Hdm2/HdmX antagonists. The potential therapeutic use of these peptides, however, is limited by their poor stability and bioavailability. Here, we report the engineering of the cyclotide MCoTI-I to efficiently antagonize intracellular p53 degradation. The resulting cyclotide MCo-PMI was able to bind with low nanomolar affinity to both Hdm2 and HdmX, showed high stability in human serum, and was cytotoxic to wild-type p53 cancer cell lines by activating the p53 tumor suppressor pathway both in vitro and in vivo. These features make the cyclotide MCoTI-I an optimal scaffold for targeting intracellular protein-protein interactions.

In Vivo Activation of Azipropofol Prolongs Anesthesia and Reveals Synaptic Targets

General anesthetic photolabels have been instrumental in discovering and confirming protein binding partners and binding sites of these promiscuous ligands. We report the in vivo photoactivation of meta-azipropofol, a potent analog of propofol, in Xenopus laevis tadpoles. Covalent adduction of meta-azipropofol in vivo prolongs the primary pharmacologic effect of general anesthetics in a behavioral phenotype we termed "optoanesthesia." Coupling this behavior with a tritiated probe, we performed unbiased, time-resolved gel proteomics to identify neuronal targets of meta-azipropofol in vivo. We have identified synaptic binding partners, such as synaptosomal-associated protein 25, as well as voltage-dependent anion channels as potential facilitators of the general anesthetic state. Pairing behavioral phenotypes elicited by the activation of efficacious photolabels in vivo with time-resolved proteomics provides a novel approach to investigate molecular mechanisms of general anesthetics.

In Vivo Activation of Anticipatory vs. Post-Event Autobiographical Images and Memories in Social Anxiety

The features of negative mental images and associated autobiographical memories were compared in high (n = 39) versus low (n = 46) socially anxious (HSA and LSA) participants using a modified version of the Waterloo Images and Memories Interview (WIMI; Moscovitch, Gavric, Bielak, Merrifield, & Moscovitch, 2011) either in anticipation of a videotaped speech or during post-event processing. Results indicated that negative images and memories were endorsed more frequently by HSA (vs. LSA) participants, and by those in the Anticipation (vs. Post-Event Processing) condition. Moreover, HSA relative to LSA participants reported more negative emotional consequences associated with bringing their images and memories to mind. Finally, post-event relative to anticipatory images and memories were associated with greater reported negative impact on participants' perceptions of self and others. Results and their potential theoretical and clinical implications are discussed in relation to contemporary cognitive-behavioral models of social anxiety.

Protein C Concentrate Controls Leukocyte Recruitment during Inflammation and Improves Survival during Endotoxemia after Efficient in Vivo Activation

Anti-inflammatory properties of protein C (PC) concentrate are poorly studied compared to activated protein C, although PC is suggested to be safer in clinical use. We investigated how PC interferes with the leukocyte recruitment cascade during acute inflammation and its efficacy during murine endotoxemia. We found that similar to activated protein infusion, intravenous PC application reduced leukocyte recruitment in inflamed tissues in a dose- and time-dependent manner. During both tumor necrosis factor-α induced and trauma-induced inflammation of the cremaster muscle, intravital microscopy revealed that leukocyte adhesion and transmigration, but not rolling, were profoundly inhibited by 100 U/kg PC. Moreover, PC blocked leukocyte emigration into the bronchoalveolar space during lipopolysaccharide (LPS) induced acute lung injury. PC was efficiently activated in a murine endotoxemia model, which reduced leukocyte infiltration of organs and strongly improved survival (75% versus 25% of control mice). Dependent on the inflammatory model, PC provoked a significant inhibition of leukocyte recruitment as early as 1 hour after administration. PC-induced inhibition of leukocyte recruitment during acute inflammation critically involves thrombomodulin-mediated PC activation, subsequent endothelial PC receptor and protease-activated receptor-1-dependent signaling, and down-regulation of intercellular adhesion molecule 1 leading to reduced endothelial inflammatory response. We conclude that during acute inflammation and sepsis, PC is a fast acting and effective therapeutic approach to block leukocyte recruitment and improve survival.

NADPH Oxidase NOX5-S Mediates Acid-Induced DNA Damage in Barrett's Esophageal Adenocarcinoma Cells

G A A b st ra ct s study we examine the role of mPGES1 in the acid-induced increase in PGE2 production and cell proliferation, and studied the role of NADPH oxidase NOX5-S in acid-induced upregulation of mPGES1 in an EA cell line FLO. RT-PCR shows that mPGES1, mPGES2 and cytosolic PGES (cPGES) are present in FLO cells. Pulsed acid treatment increased mPGES1 mRNA by 110% and mPGES2 by 10%, but did not have any effect on cPGES mRNA. Acid treatment significantly increased mPGES1 protein expression. Knockdown of mPGES1 by mPGES1 siRNA blocked acid-induced increase in PGE2 production and thymidine incorporation (an indicator of cell proliferation rate). Knockdown of NOX5-S by NOX5 siRNA significantly inhibited acid-induced increase in mPGES1 expression, PGE2 production and thymidine incorporation. Overexpression of NOX5-S significantly increased the luciferase activity in FLO cells transfected with a NF-κB In Vivo activation reporter plasmid pNF-κBLuc. Knockdown of NF-κB1 p50 by p50 siRNA almost abolished acid-induced increase in mPGES1 expression, PGE2 production and thymidine incorporation. In a chromatin immunoprecipitation assay, the promoter region of mPGES1 DNA was detectable in the immunoprecipitated chromatin sample of FLO cell lysate with a p50 antibody, indicating that p50 binds to mPGES1 promoter. We conclude that mPGES1 mediates acid-induced increase in PGE2 production and cell proliferation. Acid-induced mPGES1 expression depends on activation of NOX5-S and NF-κB1 p50. Microsomal PGES1 may be a potential target to prevent or treat EA. Supported by NIH NIDDK R01 DK080703.

In Vivo Activation of the Intracrine Vitamin D Pathway in Innate Immune Cells and Mammary Tissue during a Bacterial Infection

Numerous in vitro studies have shown that toll-like receptor signaling induces 25-hydroxyvitamin D3 1α-hydroxylase (1α-OHase; CYP27B1) expression in macrophages from various species. 1α-OHase is the primary enzyme that converts 25-hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Subsequently, synthesis of 1,25(OH)2D3 by 1α-OHase in macrophages has been shown to modulate innate immune responses of macrophages. Despite the numerous in vitro studies that have shown 1α-OHase expression is induced in macrophages, however, evidence that 1α-OHase expression is induced by pathogens in vivo is limited. The objective of this study was to evaluate 1α-OHase gene expression in macrophages and mammary tissue during an in vivo bacterial infection with Streptococcus uberis. In tissue and secreted cells from the infected mammary glands, 1α-OHase gene expression was significantly increased compared to expression in tissue and cells from the healthy mammary tissue. Separation of the cells by FACS9 revealed that 1α-OHase was predominantly expressed in the CD14+ cells isolated from the infected mammary tissue. The 24-hydroxylase gene, a gene that is highly upregulated by 1,25(OH)2D3, was significantly more expressed in tissue and cells from the infected mammary tissue than from the healthy uninfected mammary tissue thus indicating significant local 1,25(OH)2D3 production at the infection site. In conclusion, this study provides the first in vivo evidence that 1α-OHase expression is upregulated in macrophages in response to bacterial infection and that 1α-OHase at the site of infection provides 1,25(OH)2D3 for local regulation of vitamin D responsive genes.

1 CGMP Signaling Regulates Liver Sinusoidal Endothelial Cell (SEC) Phenotype and Accelerates Reversal of Cirrhosis

Differentiated, but not capillarized, SEC promote reversion of stellate cells (HSC) to quiescence. Capillarization precedes fibrosis In Vivo. Thus restoring SEC differentiation may promote resolution of fibrosis. This study examines whether NO maintains SEC phenotype In Vitro through the soluble guanylate cyclase (sGC)/cGMP/protein kinase G (PKG) pathway and whether In Vivo activation of sGC accelerates reversal of capillarization and cirrhosis. Methods Rat SEC were cultured for 2 days with inhibitors or agonists and examined by scanning EM for fenestrae in sieve plates, defining features of SEC differentiation. Controls: SEC cultured alone (complete defenestration) or with 40 ng/ml VEGF (fully fenestrated). Thioacetamide-induced (200mg/kg i.p. biw for 3 weeks) cirrhosis was followed by 0.3 mg/ kg p.o. BAY 60-2770 (kind gift from Bayer Schering Pharma), an NO-independent activator of sGC. Sirius red staining for fibrosis was assessed blindly (semi-quantitative score 0-6). In Vitro studies 1.SEC cultured with VEGF plus L-NAME (nitric oxide synthase inhibitor), with VEGF/ODQ (sGC inhibitor) or with VEGF/Rp-8-pCPT-PET-cGMPS (PKG inhibitor) defenestrated completely. Thus the NO/cGMP pathway is necessary to maintain fenestrated SEC. 2.SEC grown with DETA-NONOate (6μM) produced similar levels of NO and cGMP to SEC cultured with VEGF, but SEC defenestrated. SEC grown with YC-1 (sGC activator) or 8-pCPT-cGMP (cGMP analog) defenestrated. Thus the NO/cGMP pathway alone is insufficient to maintain SEC phenotype. 3.SEC grown with VEGF, L-NAME and DETA-NONOate, with VEGF, L-NAME and YC-1, or with VEGF, ODQ and 8-pCPT-cGMP remained fully fenestrated. Thus maintenance of SEC phenotype requires both VEGF and the NO/cGMP pathway. 4.BAY60-2770 alone was not sufficient to reverse HSC activation, but reversal required co-cultured SEC and was NO-independent. In Vivo studies with BAY 60-2770 1-week treatment normalized cGMP level in SEC [0.82± 0.02 fmol/μg protein in normal vs 0.83± 0.05 fmol/μg protein in treated vs 0.47±0.06 fmol/μg protein in solvent controls (p= 0.011)] and completely reversed capillarization [normal porosity 6.80 ± 0.31%; 6.24±0.37% in treated vs 3.14 ± 0.22% in solvent controls (p<0.01)], but cirrhosis persisted. 2-week treatment caused reversal to bridging fibrosis vs persistent cirrhosis in solvent controls. Conclusions Both VEGF independent of NO plus VEGF-stimulated NO acting through the sGC/cGMP/PKG pathway are necessary and sufficient to maintain SEC differentiation. Reversal of HSC activation by sGC activation requires the presence of SEC. Stimulation of sGC reversed capillarization, which promoted reversal of cirrhosis.