Pentraxin-3 modulates lipopolysaccharide-induced inflammatory response and attenuates liver injury.

Acute-on-chronic liver injury is characterized by an important inflammatory response frequently associated with endotoxemia. In this context, acute-phase proteins such as Pentraxin-3 (PTX3) are released; however, little is known about their role in chronic liver disease. The aim of this study was to elucidate the role of PTX3 in liver injury. The role of PTX3 was evaluated in cultured human cells, liver tissue slices, and mice with acute-on-chronic liver injury. PTX3 expression was assessed in tissue and serum samples from 54 patients with alcoholic hepatitis. PTX3 expression was up-regulated in animal models of liver injury and strongly induced by lipopolysaccharide (LPS). Liver cell fractionation showed that macrophages and activated hepatic stellate cells were the main cell types expressing PTX3 in liver injury. Ex vivo and in vivo studies showed that PTX3 treatment attenuated LPS-induced liver injury, inflammation, and cell recruitment. Mechanistically, PTX3 mediated the hepatic stellate cell wound-healing response. Moreover, PTX3 modulated LPS-induced inflammation in human primary liver macrophages and peripheral monocytes by enhancing a TIR domain-containing adapter-inducing interferon-dependent response and favoring a macrophage interleukin-10-like phenotype. Additionally, hepatic and plasma PTX3 levels were increased in patients with alcoholic hepatitis, a prototypic acute-on-chronic condition; and its expression correlated with disease severity scores, endotoxemia, infections, and short-term mortality, thus suggesting that expression of PTX3 found in patients could be a counterregulatory response to injury. CONCLUSION: Experimental and human evidence suggests that, in addition to being a potential biomarker for alcoholic hepatitis, PTX3 participates in the wound-healing response and attenuates LPS-induced liver injury and inflammation; therefore, administration of PTX3 could be a promising therapeutic strategy in acute-on-chronic conditions, particularly those associated with endotoxemia. (Hepatology 2017;66:953-968).

Pentraxin 3 promotes cardiac differentiation of mouse embryonic stem cells through JNK signaling pathway.

Cardiovascular disease is a leading cause of death worldwide, requiring the development of new therapeutic strategies including stem cell therapy. Pentraxins (PTXs) are a superfamily of proteins highly involved in different myocardial disorders, and thus this study aimed to identify the modulation of long pentraxin 3 (PTX3) in the differentiation of mouse embryonic stem cells (mESCs) toward cardiomyocytes. Cell toxicity of PTX3 was detected by MTT and LDH assays in mESCs. Embryoid bodies (EBs) were differentiated using hanging drop method, and the beating was observed under microscope. Expressional levels of early cardiac progenitor marker genes were assessed by qRT-PCR. Expression of marker proteins in early myocardial development and the activation of JNK signaling pathway was evaluated by Western blot. PTX3 treatment at 50 ng/mL significantly promoted the expression of cardiac-specific marker genes including Nkx2.5, Mef2c, Tbx5, dHand, and αMHC, and increased the expression of cardiac maturity indicative markers including connexin 43 and troponin C1. PTX3 enhanced the phosphorylation of JNK across the incubation duration, whereas the activation of p38 remained the same as control group. Co-treatment of JNK signaling pathway inhibitor SP600125 impaired the PTX3-promoted transcription of Nkx2.5, Mef2c, Tbx5, dHand, and αMHC. This study revealed the promotion of PTX3 in the differentiation of mESCs into cardiomyocytes and the underlying mechanism.

Neuronal and Peripheral Pentraxins Modify Glutamate Release and may Interact in Blood-Brain Barrier Failure.

Neuronal pentraxin 1 (NPTX1) has been implicated in Alzheimer's disease, being present in and around dystrophic neurons in plaques, affecting glutamatergic transmission postsynaptically and mediating effects of amyloidß. Here, we confirm the presence of NPTX1 around plaques in postmortem Alzheimer's disease brain and report that acutely applied human NPTX1 increases paired-pulse ratio at mouse CA3-CA1 hippocampal synapses, indicating a decrease in glutamate release. In contrast, chronic exposure to NPTX1, NPTX2, or NPTX receptor decreases paired-pulse ratio, mimicking some of the earliest changes in mice expressing familial Alzheimer's disease genes. The peripheral pentraxin, serum amyloid P component (SAP), causes similar synaptic effects to NPTX1. The presence of SAP on amyloid plaques in Alzheimer's disease confirms that it can enter the brain. We show that SAP and neuronal pentraxins can interact and that SAP can enter the brain if the blood-brain barrier is compromised, suggesting that peripheral pentraxins could affect central synaptic transmission via this interaction, especially in the event of blood-brain barrier breakdown.

Effect of Recombinant Human Pentraxin 2 vs Placebo on Change in Forced Vital Capacity in Patients With Idiopathic Pulmonary Fibrosis: A Randomized Clinical Trial.

Importance: Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease with poor prognosis. Approved therapies do not halt disease progression. Objective: To determine the effect of recombinant human pentraxin 2 vs placebo on change from baseline to week 28 in mean forced vital capacity (FVC) percentage of predicted value. Design, Setting, and Participants: Phase 2, randomized, double-blind, placebo-controlled trial conducted at 18 sites in 7 countries of eligible patients with IPF (N = 117; aged 40-80 years; FVC ≥50% and ≤90% predicted; ratio of forced expiratory volume in the first second/FVC >0.70; diffusing capacity for carbon monoxide [Dlco] ≥25% and ≤90% predicted; and distance of ≥150 m on the 6-minute walk test). Study period was August 2015-May 2017. Interventions: Patients were randomized to receive either recombinant human pentraxin 2 (10 mg/kg intravenous every 4 weeks, n = 77) or placebo (n = 39) for 24 weeks, and stratified by concurrent IPF treatment status. Main Outcomes and Measures: The primary end point was the least-squares mean change in FVC percentage of predicted value from baseline to week 28 (minimal clinically important difference, decline of 2%-6%). Secondary end points included mean change in lung volumes (total, normal, and interstitial lung abnormalities) on high-resolution computed tomography (HRCT) and 6-minute walk distance (minimal clinically important difference, 24-45 m). Results: Of 117 randomized patients, 116 received at least 1 dose of study drug (mean age, 68.6 years; 81.0% men; mean time since IPF diagnosis, 3.8 years), and 111 (95.7%) completed the study. The least-squares mean change in FVC percentage of predicted value from baseline to week 28 in patients treated with recombinant human pentraxin 2 was -2.5 vs -4.8 for those in the placebo group (difference, +2.3 [90% CI, 1.1 to 3.5]; P = .001). No significant treatment differences were observed in total lung volume (difference, 93.5 mL [90% CI, -27.7 to 214.7]), quantitative parenchymal features on HRCT (normal lung volume difference, -1.2% [90% CI, -4.4 to 1.9]; interstitial lung abnormalities difference, 1.1% [90% CI, -2.2 to 4.3]), or measurement of Dlco (difference, -0.4 [90% CI, -2.6 to 1.7]). The change in 6-minute walk distance was -0.5 m for patients treated with recombinant human pentraxin 2 vs -31.8 m for those in the placebo group (difference, +31.3 m [90% CI, 17.4 to 45.1]; P < .001). The most common adverse events in the recombinant human pentraxin 2 vs placebo group were cough (18% vs 5%), fatigue (17% vs 10%), and nasopharyngitis (16% vs 23%). Conclusions and Relevance: In this preliminary study, recombinant human pentraxin 2 vs placebo resulted in a slower decline in lung function over 28 weeks for patients with idiopathic pulmonary fibrosis. Further research should more fully assess efficacy and safety. Trial Registration: clinicaltrials.gov Identifier: NCT02550873.

C-reactive protein (CRP) but not the related pentraxins serum amyloid P and PTX3 inhibits the proliferation and induces apoptosis of the leukemia cell line Mono Mac 6.

BACKGROUND: Pentraxins are a family of highly conserved secreted proteins that regulate the innate immune system, including monocytes and macrophages. C-reactive protein (CRP) is a plasma protein whose levels can rise to 1000 µg/ml from the normal <3 µg/ ml during inflammation. RESULTS: We find that CRP inhibits proliferation of the human myeloid leukemia cell line Mono Mac 6 with an IC50 of 75 µg/ ml by inducing apoptosis of these cells. The related proteins serum amyloid P (SAP) and pentraxin 3 (PTX3) do not inhibit Mono Mac 6 proliferation. CRP has no significant effect on the proliferation of other leukemia cell lines such as HL-60, Mono Mac 1, K562, U937, or THP-1, or the survival of normal peripheral blood cells. The effect of CRP appears to be dependent on the CRP receptor FcγRI, and is negatively regulated by a phosphatidylinositol -3-kinase pathway. CONCLUSION: These data reveal differential signaling by pentraxins on immune cells, and suggest that CRP can regulate the proliferation of some myeloid leukemia cells.

Pentraxin 3 Activates JNK Signaling and Regulates the Epithelial-To-Mesenchymal Transition in Renal Fibrosis.

BACKGROUND/AIMS: Tubulointerstitial fibrosis can lead to end-stage renal disease. Pentraxin 3 (PTX3) is an acute phase protein produced by resident and innate immunity cells. We investigated the effect of PTX3 on cultured human proximal tubular epithelial (HK-2) cells and a rat unilateral ureteral obstruction (UUO) model of renal fibrosis. METHODS: Gain-of-function experiments were used to examine the effect of recombinant human PTX3 (Rh-PTX3) on HK-2 cells. Cell proliferation (MTT assay) and in vitro cell migration were measured. The levels of PTX3, p-JNK, and EMT markers were measured using immunohistochemistry, RT-PCR, and western blotting in UUO rats and HK-2 cells. RESULTS: HK-2 cells treated with Rh PTX3 did not affect cell viability, but significantly increased cell migration. Moreover, Rh-PTX3 increased the expression of snail, slug, N-cadherin, and vimentin, decreased the expression of E-cadherin, and increased the phosphorylation of JNK. SP600126 (a specific JNK inhibitor) enhanced the effects of Rh-PTX3. Rats with UUO exhibited time-dependent increased levels of PTX3, p-JNK, and vimentin, and decreased expression of E-cadherin. CONCLUSIONS: Our results suggest that PTX3 induces cell migration via upregulation of EMT in a JNK-dependent mechanism, and highlight the role of PTX3 in the pathogenesis renal fibrosis.

A single amino acid substitution in the hemagglutinin of H3N2 subtype influenza A viruses is associated with resistance to the long pentraxin PTX3 and enhanced virulence in mice.

The long pentraxin, pentraxin 3 (PTX3), can play beneficial or detrimental roles during infection and disease by modulating various aspects of the immune system. There is growing evidence to suggest that PTX3 can mediate antiviral activity in vitro and in vivo. Previous studies demonstrated that PTX3 and the short pentraxin serum amyloid P express sialic acids that are recognized by the hemagglutinin (HA) glycoprotein of certain influenza A viruses (IAV), resulting in virus neutralization and anti-IAV activity. In this study, we demonstrate that specificity of both HA and the viral neuraminidase for particular sialic acid linkages determines the susceptibility of H1N1, H3N2, and H7N9 strains to the antiviral activities of PTX3 and serum amyloid P. Selection of H3N2 virus mutants resistant to PTX3 allowed for identification of amino acid residues in the vicinity of the receptor-binding pocket of HA that are critical determinants of sensitivity to PTX3; this was supported by sequence analysis of a range of H3N2 strains that were sensitive or resistant to PTX3. In a mouse model of infection, the enhanced virulence of PTX3-resistant mutants was associated with increased virus replication and elevated levels of proinflammatory cytokines in the airways, leading to pulmonary inflammation and lung injury. Together, these studies identify determinants in the viral HA that can be associated with sensitivity to the antiviral activities of PTX3 and highlight its importance in the control of IAV infection.

Antibacterial effect of porcine PTX3 against Streptococcus suis type 2 infection.

Pentraxin 3 (PTX3), a soluble pattern recognition receptor, plays an important role in innate immunity and has been implicated to be a candidate resistance gene against Streptococcus suis 2 infection. To discover the antibacterial effect of porcine PTX3 against S. suis 2, the 42-kDa PTX3 protein was expressed by Chinese hamster ovary cells (CHO), and an additional eukaryotic expression vector pVAX-ptx3 was constructed. The expressed porcine PTX3 mediated a range of antibacterial activities including increasing phagocytic capacity of primary porcine alveolar macrophages (PAM) against S. suis 2 and inhibiting adhesion of S. suis 2 to human epidermoid cancer cells (Hep-2). In mouse model, pre-intramuscular injecting with pVAX-ptx3 reduced mortality and reduced bacteria loads in blood, spleen, lung and brain compared with that of control group during 2-12 h following intraperitoneal injection (i.p.) with S. suis 2. Meanwhile, the expressions of IL-6 and IL-8 in blood were increased in pVAX-ptx3 group, whereas no obvious changes about IL-10. In piglet model, bacteria load in blood of pVAX-ptx3 group was significantly lower than that of control group after i.p. with S. suis 2, correspondingly, expression of IL-6 and IL-8 were significantly increased in pVAX-ptx3 group. In contrast, white blood cell (WBC) and neutrophil cell (NEU) count of peripheral blood in pVAX-ptx3 group were lower than that of control group. These studies described a novel antibacterial role for porcine PTX3 against S. suis 2 both in vitro and in vivo and suggested that porcine PTX3 may be a potential biological agent against S. suis 2 in pig and be used for the clinical prevention and treatment of streptococcosis caused by S. suis 2.

Protection of human podocytes from shiga toxin 2-induced phosphorylation of mitogen-activated protein kinases and apoptosis by human serum amyloid P component.

Hemolytic uremic syndrome (HUS) is mainly induced by Shiga toxin 2 (Stx2)-producing Escherichia coli. Proteinuria can occur in the early phase of the disease, and its persistence determines the renal prognosis. Stx2 may injure podocytes and induce proteinuria. Human serum amyloid P component (SAP), a member of the pentraxin family, has been shown to protect against Stx2-induced lethality in mice in vivo, presumably by specific binding to the toxin. We therefore tested the hypothesis that SAP can protect against Stx2-induced injury of human podocytes. To elucidate the mechanisms underlying podocyte injury in HUS-associated proteinuria, we assessed Stx2-induced activation of mitogen-activated protein kinases (MAPKs) and apoptosis in immortalized human podocytes and evaluated the impact of SAP on Stx2-induced damage. Human podocytes express Stx2-binding globotriaosylceramide 3. Stx2 applied to cultured podocytes was internalized and then activated p38α MAPK and c-Jun N-terminal kinase (JNK), important signaling steps in cell differentiation and apoptosis. Stx2 also activated caspase 3, resulting in an increased level of apoptosis. Coincubation of podocytes with SAP and Stx2 mitigated the effects of Stx2 and induced upregulation of antiapoptotic Bcl2. These data suggest that podocytes are a target of Stx2 and that SAP protects podocytes against Stx2-induced injury. SAP may therefore be a useful therapeutic option.

Efficacy of PTX3 and posaconazole combination in a rat model of invasive pulmonary aspergillosis.

Posaconazole is currently used for the prophylaxis of invasive pulmonary aspergillosis (IPA). Limitations to posaconazole usage are drug-drug interactions and side effects. PTX3 is an innate immunity glycoprotein with opsonic activity, proven to be protective in IPA animal models. This study investigated the combination of posaconazole with PTX3. The results indicate synergy between PTX3 and posaconazole against aspergillosis, suggesting that a combination of reduced doses of posaconazole with the immune response enhancer PTX3 might represent a treatment option with a higher therapeutic index than posaconazole.

Long pentraxin-3 as an epithelial-stromal fibroblast growth factor-targeting inhibitor in prostate cancer.

Fibroblast growth factors (FGFs) exert autocrine/paracrine functions in prostate cancer by stimulating angiogenesis and tumour growth. Here dihydrotestosterone (DHT) up-regulates FGF2 and FGF8b production in murine TRAMP-C2 prostate cancer cells, activating a FGF-dependent autocrine loop of stimulation. The soluble pattern recognition receptor long pentraxin-3 (PTX3) acts as a natural FGF antagonist that binds FGF2 and FGF8b via its N-terminal domain. We demonstrate that recombinant PTX3 protein and the PTX3-derived pentapeptide Ac-ARPCA-NH2 abolish the mitogenic response of murine TRAMP-C2 cells and human LNCaP prostate cancer cells to DHT and FGFs. Also, PTX3 hampers the angiogenic activity of DHT-activated TRAMP-C2 cells on the chick embryo chorioallantoic membrane (CAM). Accordingly, human PTX3 overexpression inhibits the mitogenic activity exerted by DHT or FGFs on hPTX3_TRAMP-C2 cell transfectants and their angiogenic activity. Also, hPTX3_TRAMP-C2 cells show a dramatic decrease of their angiogenic and tumourigenic potential when grafted in syngeneic or immunodeficient athymic male mice. A similar inhibitory effect is observed when TRAMP-C2 cells overexpress only the FGF-binding N-terminal PTX3 domain. In keeping with the anti-tumour activity of PTX3 in experimental prostate cancer, immunohistochemical analysis of prostate needle biopsies from primary prostate adenocarcinoma patients shows that parenchymal PTX3 expression, abundant in basal cells of normal glands, is lost in high-grade prostatic intraepithelial neoplasia and in invasive tumour areas. These results identify PTX3 as a potent FGF antagonist endowed with anti-angiogenic and anti-neoplastic activity in prostate cancer.

Fibroblast growth factor 2 involved in the pathogenesis of synovial chondromatosis of temporomandibular joint.

BACKGROUND: Synovial chondromatosis (SC) of temporomandibular joint (TMJ) is a rare proliferative disorder characterized by the formation of cartilaginous or osteocartilaginous nodules in synovium and joint space. Fibroblast growth factor 2 (FGF-2) is frequently applied in chondrogenic differentiation assays. Therefore, we hypothesized that FGF-2 might involved in the pathogenesis of SC. METHODS: SC synovium and loose bodies (LBs) specimens were observed by histological and immunohistochemical methods. Real-time PCR was conducted for comparing genes expressions in SC and normal synovium. SC synoviocytes were stimulated by FGF-2 in the presence or absence of its antagonist long pentraxin-3 (PTX3) for 6 days. Real-time PCR and alkaline phosphatase (ALP) activity were performed to examine the effects exerted by FGF-2 and PTX3. RESULTS: SC synovium, no matter facing the articular cavity or covering LB, was characterized by increased quantity of synoviocytes and blood vessels. FGF-2 was expressed in chondrocytes and fibroblast-like cells of LBs, and the wall of blood vessels. Expressions of chondrogenic genes (Sox9 and Wnt-4), osteogenic genes (Foxc2), FGF-2, and VEGF-A mRNA were significantly higher in SC synovium than that of the control group. The stimulation of FGF-2 on SC synoviocytes increased ALP activity and expressions of chondrogenic genes (Sox9, Col2α1, and Aggrecan), osteogenic genes (Foxc2, osteocalcin, and Col1α1), and VEGF-A, but PTX3 inhibited these effects. CONCLUSION: FGF-2 was responsible for the formation of cartilaginous loose bodies and involved in the pathogenesis of SC.

Macrophage differentiation and polarization via phosphatidylinositol 3-kinase/Akt-ERK signaling pathway conferred by serum amyloid P component.

Macrophage differentiation and polarization is influenced by, and act on, many processes associated with autoimmunity. However, the molecular mechanisms underlying macrophage polarization in systemic lupus erythematosus (SLE) remain largely debated. We previously demonstrated that macrophage M2b polarization conferred by activated lymphocyte-derived (ALD)-DNA immunization could initiate and propagate murine lupus nephritis. Serum amyloid P component (SAP), a conserved acute-phase protein in mice, has been reported to bind to DNA and modulate immune responses. In this study, murine SAP was shown to promote macrophage-mediated ALD-DNA uptake through binding to ALD-DNA (SAP/ALD-DNA). Moreover, macrophage phenotypic switch from a proinflammatory M2b phenotype induced by ALD-DNA alone to an anti-inflammatory M2a phenotype stimulated with SAP/ALD-DNA were found because of PI3K/Akt-ERK signaling activation. Both in vivo SAP supplements and adoptive transfer of ex vivo programmed M2a macrophages induced by SAP/ALD-DNA into SLE mice could efficiently alleviate lupus nephritis. Importantly, increased IL-10 secretion, accompanied by anti-inflammatory effect exerted by M2a macrophages, was found to predominantly impede macrophage M2b polarization. Furthermore, neutralization of IL-10 notably reduced the suppressive effect of M2a macrophages. Our results demonstrate that binding of SAP to ALD-DNA could switch macrophage phenotypic polarization from proinflammatory M2b to anti-inflammatory M2a via PI3K/Akt-ERK signaling activation, thus exerting protective and therapeutic interventions on murine lupus nephritis. These data provide a possible molecular mechanism responsible for modulation of macrophage polarization in the context of lupus nephritis and open a new potential therapeutic avenue for SLE.

SAP suppresses the development of experimental autoimmune encephalomyelitis in C57BL/6 mice.

Experimental autoimmune encephalomyelitis (EAE) is a CD4(+) T cell-mediated disease of the central nervous system. Serum amyloid P component (SAP) is a highly conserved plasma protein named for its universal presence in amyloid deposits. Here we report that SAP-transgenic mice had unexpectedly attenuated EAE due to impaired encephalitogenic responses. Following induction with myelin oligodendroglial glycoprotein (MOG) peptide 35-55 in complete Freund's adjuvant, SAP-transgenic mice showed reduced spinal cord inflammation with lower severity of EAE attacks as compared with control C57BL/6 mice. However, in SAP-Knockout mice, the severity of EAE is enhanced. Adoptive transfer of Ag-restimulated T cells from wild type to SAP-transgenic mice, or transfer of SAP-transgenic Ag-restimulated T cells to control mice, induced milder EAE. T cells from MOG-primed SAP-transgenic mice showed weak proliferative responses. Furthermore, in SAP-transgenic mice, there is little infiltration of CD45-positive cells in the spinal cord. In vitro, SAP suppressed the secretion of interleukin-2 stimulated by P-selectin and blocked P-selectin binding to T cells. Moreover, SAP could change the affinity between α4-integrin and T cells. These data suggested that SAP could antagonize the development of the acute phase of inflammation accompanying EAE by modulating the function of P-selectin.

Serum amyloid P attenuates M2 macrophage activation and protects against fungal spore-induced allergic airway disease.

BACKGROUND: Aspergillus fumigatus conidia aggravate asthmatic responses. Lung macrophages normally kill fungal conidia, but the presence of type 2 cytokines during asthma contributes to the alternative (or M2) activation of these cells, which secrete proallergic factors and exhibit impaired innate immunity. OBJECTIVE: Considering that pentraxins modulate macrophage function, we examined the effect of C-reactive protein (CRP) and serum amyloid P (SAP) in an experimental model of A fumigatus-induced allergic airway disease. METHODS: The effects of SAP and CRP on M2 macrophage differentiation were examined in vitro, and the in vivo effects of these pentraxins were analyzed in the asthma model. RESULTS: SAP inhibited the generation of M2 markers, such as arginase and the chitinase Ym-1, through an FcγR-dependent mechanism in cultured macrophages. This effect correlated with a decrease in signal transducer and activator of transcription 6 (STAT6) phosphorylation in SAP-treated M2 macrophages. In vivo treatment with SAP significantly decreased methacholine-induced bronchial resistance, mucus cell metaplasia, the number of "found in inflammatory zone 1" (FIZZ1)-positive cells in the lungs, and collagen deposition compared with the control group. CRP had a modest effect on M2 differentiation, and in vivo treatment with CRP had a minor effect or exacerbated A fumigatus-induced lung disease. Finally, the adoptive transfer of SAP-pretreated M2 macrophages into allergic mice significantly attenuated disease when compared with nontransferred or M2-transferred control groups. CONCLUSIONS: These findings demonstrate that SAP is a potent inhibitor of M2 macrophage differentiation and represents a novel therapy in A fumigatus-induced allergic disease.

The Protective Role of the Long Pentraxin PTX3 in Spontaneously Hypertensive Rats with Heart Failure.

Pentraxin 3 (PTX3) is synthesized locally and released into the circulation, reflecting local inflammation in the cardiovascular system. Therefore, we conducted a study to explore the effect of PTX3 in spontaneously hypertensive heart failure (SHHF) rats. Sprague Dawley (SD) and SHHF rats were treated with recombinant PTX3 protein, and the blood pressure (BP) and echocardiographic parameters were collected. Radioimmunoassay, enzyme immunoassay and enzyme-linked immunosorbent assay (ELISA) were applied to detect plasma levels of atrial/B-type natriuretic peptide (ANP/BNP) and PTX3. The pathological changes in the myocardial tissues were observed by hematoxylin and eosin (HE) and Masson stainings. The mRNA and protein expressions were detected by quantitative real-time reverse-transcription polymerase chain reaction (qPCR) and western blotting. Cardiomyocyte apoptosis was evaluated by TUNEL staining and DNA fragmentation test. Increased plasma concentrations of PTX3 were found in SHHF rats compared with SD rats, which was further enhanced by recombinant PTX3 protein. After injection with recombinant PTX3 protein, the heart function was improved in SHHF rats with the decreased systolic and diastolic BP, and the reduced plasma levels of ANP and BNP. Moreover, PTX3 improved the myocardial damage and interstitial fibrosis in SHHF rats with reduced cardiomyocyte apoptosis and decreased mRNA expressions of pro-inflammatory factors in myocardial tissues. PTX3 could decrease the BP and plasma levels of ANP and BNP in SHHF rats, as well as improve the inflammation, cardiomyocyte apoptosis, and pathological changes of myocardial tissues, suggesting it may be a useful intervention in the treatment of SHHF.

Reduced Sialylation and Bioactivity of the Antifibrotic Protein Serum Amyloid P in the Sera of Patients with Idiopathic Pulmonary Fibrosis.

Pulmonary fibrosis is a chronic and generally fatal disorder characterized by progressive formation of scar-like tissue in the lungs. Sialic acids are often found as the terminal sugar on extracellular glycoconjugates such as protein glycosylations. Sialidases, also known as neuraminidases, desialylate glycoconjugates. Serum amyloid P (SAP), a pentameric serum glycoprotein that has two sialic acids on each polypeptide, inhibits the differentiation of monocytes into fibrocytes and promotes human PBMCs to accumulate high extracellular levels of IL-10. When SAP is desialylated with sialidase, the effects of SAP on fibrocyte differentiation and IL-10 accumulation are strongly inhibited. Intriguingly, in patients with pulmonary fibrosis, there are increased levels of sialidase activity in the bronchoalveolar lavage fluid, increased levels of sialidases in the lungs, and decreased levels of SAP in the sera. To elucidate the role of SAP desialylation in idiopathic pulmonary fibrosis (IPF) pathogenesis, we purified SAP from the serum of IPF patients and healthy controls and measured the extent of sialylation and bioactivity of the purified SAP. We find that some IPF patients have abnormally high levels of the sialidase NEU3 in their sera and that the SAP in the sera of IPF patients has an abnormally high extent of desialylation and an abnormally low ability to inhibit fibrocyte differentiation and induce extracellular IL-10 accumulation by PBMC. These results suggest that SAP desialylation may play a role in IPF pathogenesis and that inhibiting NEU3 could be a potential therapeutic target for IPF.

Pivotal Advance: Th-1 cytokines inhibit, and Th-2 cytokines promote fibrocyte differentiation.

CD14+ peripheral blood monocytes can differentiate into fibroblast-like cells called fibrocytes, which are associated with and are at least partially responsible for wound healing and fibrosis in multiple organ systems. Signals regulating fibrocyte differentiation are poorly understood. In this study, we find that when added to human PBMCs cultured in serum-free medium, the profibrotic cytokines IL-4 and IL-13 promote fibrocyte differentiation without inducing fibrocyte or fibrocyte precursor proliferation. We also find that the potent, antifibrotic cytokines IFN-gamma and IL-12 inhibit fibrocyte differentiation. In our culture system, IL-1beta, IL-3, IL-6, IL-7, IL-16, GM-CSF, M-CSF, fetal liver tyrosine kinase 3, insulin growth factor 1, vascular endothelial growth factor, and TNF-alpha had no significant effect on fibrocyte differentiation. IL-4, IL-13, and IFN-gamma act directly on monocytes to regulate fibrocyte differentiation, and IL-12 acts indirectly, possibly through CD16-positive NK cells. We previously identified the plasma protein serum amyloid P (SAP) as a potent inhibitor of fibrocyte differentiation. When added together, the fibrocyte-inhibitory activity of SAP dominates the profibrocyte activities of IL-4 and IL-13. The profibrocyte activities of IL-4 and IL-13 and the fibrocyte-inhibitory activities of IFN-gamma and IL-12 counteract each other in a concentration-dependent manner. These results indicate that the complex mix of cytokines and plasma proteins present in inflammatory lesions, wounds, and fibrosis will influence fibrocyte differentiation.

Serum amyloid P inhibits dermal wound healing.

The repair of open wounds depends on granulation tissue formation and contraction, which is primarily mediated by myofibroblasts. A subset of myofibroblasts originates from bone-marrow-derived monocytes which differentiate into fibroblast-like cells called fibrocytes. Serum amyloid P (SAP) inhibits differentiation of monocytes into fibrocytes. Thus, we hypothesized that the addition of exogenous SAP would hinder the normal wound healing process. Excisional murine dorsal wounds were either injected with SAP (intradermal group) or the mice were treated with systemic SAP (intraperitoneal group) and compared with animals treated with vehicle. Grossly, SAP-treated wounds closed slower than respective controls in both groups. Histologically, the contraction rate was slower in SAP-treated wounds in both groups and the reepithelialization rate was slower in the intraperitoneal group. Furthermore, significantly less myofibroblasts expressing alpha-smooth muscle actin were noted in the intraperitoneal group wounds compared with controls. These data suggest that SAP delays normal murine dermal wound healing, probably due to increased inhibition of fibrocyte differentiation, and ultimately a decreased wound myofibroblast population. SAP may provide a potential therapeutic target to prevent or limit excessive fibrosis associated with keloid or hypertrophic scar formation. Furthermore, SAP removal from wound fluid could potentially accelerate the healing of chronic, nonhealing wounds.

Exogenous pentraxin-3 inhibits the reactive oxygen species-mitochondrial and apoptosis pathway in acute kidney injury.

Pentraxin-3 (PTX3) is a long-form member of the pentraxin family of proteins that has been studied in inflammatory diseases and in various organs. We found that PTX3 protects kidney cells during ischemia and proinflammatory acute kidney injury. The aim of this study was to develop an in vitro experimental model of acute kidney injury and to analyze the protective mechanism of exogenous recombinant PTX3. In this study, cells of the HK-2 renal tubular cell line were treated with a calcium ionophore (A23187), which induced injury by increasing intracellular calcium concentrations and inducing calpain activity and the generation of reactive oxygen species. Exposure of cells to PTX3 significantly attenuated these effects. In addition, the activity of caspase-3 and PARP-1 were decreased in ischemic cells exposed to exogenous recombinant PTX3. PTX3 stabilized the mitochondrial membrane potential and suppressed apoptosis, resulting in the protection of renal tubular cells from ischemic injury.