The role of endogenous Smad7 in regulating macrophage phenotype following myocardial infarction.

Smad7 restrains TGF-ß responses, and has been suggested to exert both pro- and anti-inflammatory actions that may involve effects on macrophages. Myocardial infarction triggers a macrophage-driven inflammatory response that not only plays a central role in cardiac repair, but also contributes to adverse remodeling and fibrosis. We hypothesized that macrophage Smad7 expression may regulate inflammation and fibrosis in the infarcted heart through suppression of TGF-ß responses, or via TGF-independent actions. In a mouse model of myocardial infarction, infiltration with Smad7+ macrophages peaked 7 days after coronary occlusion. Myeloid cell-specific Smad7 loss in mice had no effects on homeostatic functions and did not affect baseline macrophage gene expression. RNA-seq predicted that Smad7 may promote TREM1-mediated inflammation in infarct macrophages. However, these alterations in the transcriptional profile of macrophages were associated with a modest and transient reduction in infarct myofibroblast infiltration, and did not affect dysfunction, chamber dilation, scar remodeling, collagen deposition, and macrophage recruitment. In vitro, RNA-seq and PCR arrays showed that TGF-ß has profound effects on macrophage profile, attenuating pro-inflammatory cytokine/chemokine expression, modulating synthesis of matrix remodeling genes, inducing genes associated with sphingosine-1 phosphate activation and integrin signaling, and inhibiting cholesterol biosynthesis genes. However, Smad7 loss did not significantly affect TGF-ß-mediated macrophage responses, modulating synthesis of only a small fraction of TGF-ß-induced genes, including Itga5, Olfml3, and Fabp7. Our findings suggest a limited role for macrophage Smad7 in regulation of post-infarction inflammation and repair, and demonstrate that the anti-inflammatory effects of TGF-ß in macrophages are not restrained by endogenous Smad7 induction.

Differential effects of Smad2 and Smad3 in regulation of macrophage phenotype and function in the infarcted myocardium.

TGF-ßs regulate macrophage responses, by activating Smad2/3. We have previously demonstrated that macrophage-specific Smad3 stimulates phagocytosis and mediates anti-inflammatory macrophage transition in the infarcted heart. However, the role of macrophage Smad2 signaling in myocardial infarction remains unknown. We studied the role of macrophage-specific Smad2 signaling in healing mouse infarcts, and we explored the basis for the distinct effects of Smad2 and Smad3. In infarct macrophages, Smad3 activation preceded Smad2 activation. In contrast to the effects of Smad3 loss, myeloid cell-specific Smad2 disruption had no effects on mortality, ventricular dysfunction and adverse remodeling, after myocardial infarction. Macrophage Smad2 loss modestly, but transiently increased myofibroblast density in the infarct, but did not affect phagocytic removal of dead cells, macrophage infiltration, collagen deposition, and scar remodeling. In isolated macrophages, TGF-ß1, -ß2 and -ß3, activated both Smad2 and Smad3, whereas BMP6 triggered only Smad3 activation. Smad2 and Smad3 had similar patterns of nuclear translocation in response to TGF-ß1. RNA-sequencing showed that Smad3, and not Smad2, was the main mediator of transcriptional effects of TGF-ß on macrophages. Smad3 loss resulted in differential expression of genes associated with RAR/RXR signaling, cholesterol biosynthesis and lipid metabolism. In both isolated bone marrow-derived macrophages and in infarct macrophages, Smad3 mediated synthesis of Nr1d2 and Rara, two genes encoding nuclear receptors, that may be involved in regulation of their phagocytic and anti-inflammatory properties. In conclusion, the in vivo and in vitro effects of TGF-ß on macrophage function involve Smad3, and not Smad2.

Macrophage Smad3 Protects the Infarcted Heart, Stimulating Phagocytosis and Regulating Inflammation.

RATIONALE: TGF (transforming growth factor)-ß is critically involved in myocardial injury, repair, and fibrosis, activating both Smad (small mothers against decapentaplegic)-dependent and non-Smad pathways. The in vivo role of TGF-ß signaling in regulation of macrophage function is poorly understood. We hypothesized that in the infarcted myocardium, activation of TGF-ß/Smad signaling in macrophages may regulate repair and remodeling. OBJECTIVE: To investigate the role of macrophage-specific TGF-ß Smad3 signaling in a mouse model of myocardial infarction and to dissect the mechanisms mediating Smad-dependent modulation of macrophage function. METHODS AND RESULTS: TGF-ßs markedly activated Smad3 in macrophages, without affecting Smad-independent pathways. Phagocytosis rapidly and directly activated macrophage Smad3, in the absence of active TGF-ß release. MyS3KO (myeloid cell-specific Smad3 knockout) mice had no baseline defects but exhibited increased late mortality and accentuated dilative postmyocardial infarction remodeling. Adverse outcome in infarcted MyS3KO mice was associated with perturbations in phagocytic activity, defective transition of macrophages to an anti-inflammatory phenotype, scar expansion, and accentuated apoptosis of border zone cardiomyocytes. In vitro, Smad3 null macrophages exhibited reduced expression of genes associated with eat-me signals, such as Mfge8 (milk fat globule-epidermal growth factor factor 8), and reduced capacity to produce the anti-inflammatory mediators IL (interleukin)-10 and TGF-ß1, and the angiogenic growth factor VEGF (vascular endothelial growth factor). Mfge8 partly rescued the phagocytic defect of Smad3 null macrophages, without affecting inflammatory activity. Impaired anti-inflammatory actions of Smad3 null macrophages were associated with marked attenuation of phagocytosis-induced PPAR (peroxisome proliferator-activated receptor) expression. MyS3KO mice had no significant alterations in microvascular density and interstitial fibrosis in remodeling myocardial segments. CONCLUSIONS: We demonstrate that Smad3 critically regulates function of infarct macrophages, by mediating acquisition of a phagocytic phenotype and by contributing to anti-inflammatory transition. Smad3-dependent actions in macrophages protect the infarcted heart from adverse remodeling.

Protective Effects of Activated Myofibroblasts in the Pressure-Overloaded Myocardium Are Mediated Through Smad-Dependent Activation of a Matrix-Preserving Program.

RATIONALE: The heart contains abundant interstitial and perivascular fibroblasts. Traditional views suggest that, under conditions of mechanical stress, cytokines, growth factors, and neurohumoral mediators stimulate fibroblast activation, inducing ECM (extracellular matrix) protein synthesis and promoting fibrosis and diastolic dysfunction. Members of the TGF (transforming growth factor)-ß family are upregulated and activated in the remodeling myocardium and modulate phenotype and function of all myocardial cell types through activation of intracellular effector molecules, the Smads (small mothers against decapentaplegic), and through Smad-independent pathways. OBJECTIVES: To examine the role of fibroblast-specific TGF-ß/Smad3 signaling in the remodeling pressure-overloaded myocardium. METHODS AND RESULTS: We examined the effects of cell-specific Smad3 loss in activated periostin-expressing myofibroblasts using a mouse model of cardiac pressure overload, induced through transverse aortic constriction. Surprisingly, FS3KO (myofibroblast-specific Smad3 knockout) mice exhibited accelerated systolic dysfunction after pressure overload, evidenced by an early 40% reduction in ejection fraction after 7 days of transverse aortic constriction. Accelerated systolic dysfunction in pressure-overloaded FS3KO mice was associated with accentuated matrix degradation and generation of collagen-derived matrikines, accompanied by cardiomyocyte myofibrillar loss and apoptosis, and by enhanced macrophage-driven inflammation. In vitro, TGF-ß1, TGF-ß2, and TGF-ß3 stimulated a Smad3-dependent matrix-preserving phenotype in cardiac fibroblasts, suppressing MMP (matrix metalloproteinase)-3 and MMP-8 synthesis and inducing TIMP (tissue inhibitor of metalloproteinases)-1. In vivo, administration of an MMP-8 inhibitor attenuated early systolic dysfunction in pressure-overloaded FS3KO mice, suggesting that the protective effects of activated cardiac myofibroblasts in the pressure-overloaded myocardium are, at least in part, because of suppression of MMPs and activation of a matrix-preserving program. MMP-8 stimulation induces a proinflammatory phenotype in isolated macrophages. CONCLUSIONS: In the pressure-overloaded myocardium, TGF-ß/Smad3-activated cardiac fibroblasts play an important protective role, preserving the ECM network, suppressing macrophage-driven inflammation, and attenuating cardiomyocyte injury. The protective actions of the myofibroblasts are mediated, at least in part, through Smad-dependent suppression of matrix-degrading proteases.

Distinct roles of myofibroblast-specific Smad2 and Smad3 signaling in repair and remodeling of the infarcted heart.

TGF-ßs regulate fibroblast responses, by activating Smad2 or Smad3 signaling, or via Smad-independent pathways. We have previously demonstrated that myofibroblast-specific Smad3 is critically implicated in repair of the infarcted heart. However, the role of fibroblast Smad2 in myocardial infarction remains unknown. This study investigates the role of myofibroblast-specific Smad2 signaling in myocardial infarction, and explores the mechanisms responsible for the distinct effects of Smad2 and Smad3. In a mouse model of non-reperfused myocardial infarction, Smad2 activation in infarct myofibroblasts peaked 7 days after coronary occlusion. In vitro, TGF-ß1, -ß2 and -ß3, but not angiotensin 2 and bone morphogenetic proteins-2, -4 and -7, activated fibroblast Smad2. Myofibroblast-specific Smad2 and Smad3 knockout mice (FS2KO, FS3KO) and corresponding control littermates underwent non-reperfused infarction. In contrast to the increase in rupture rates and adverse remodeling in FS3KO mice, FS2KO animals had mortality comparable to Smad2 fl/fl controls, and exhibited a modest but transient improvement in dysfunction after 7 days of coronary occlusion. At the 28 day timepoint, FS2KO and Smad2 fl/fl mice had comparable adverse remodeling. Although both FS3KO and FS2KO animals had increased myofibroblast density in the infarct, only FS3KO mice exhibited impaired scar organization, associated with perturbed alignment of infarct myofibroblasts. In vitro, Smad3 but not Smad2 knockdown downmodulated fibroblast α2 and α5 integrin expression. Moreover, Smad3 knockdown reduced expression of the GTPase RhoA, whereas Smad2 knockdown markedly increased fibroblast RhoA levels. Smad3-dependent integrin expression may be important for fibroblast activation, whereas RhoA may transduce planar cell polarity pathway signals, essential for fibroblast alignment. Myofibroblast-specific Smad3, but not Smad2 is required for formation of aligned myofibroblast arrays in the infarct. The distinct in vivo effects of myofibroblast Smad2 and Smad3 may involve Smad3-dependent integrin synthesis, and contrasting effects of Smad2 and Smad3 on RhoA expression.

Opposing Actions of Fibroblast and Cardiomyocyte Smad3 Signaling in the Infarcted Myocardium.

BACKGROUND: Transforming growth factor-ßs regulate a wide range of cellular responses by activating Smad-dependent and Smad-independent cascades. In the infarcted heart, Smad3 signaling is activated in both cardiomyocytes and interstitial cells. We hypothesized that cell-specific actions of Smad3 regulate repair and remodeling in the infarcted myocardium. METHODS: To dissect cell-specific Smad3 actions in myocardial infarction, we generated mice with Smad3 loss in activated fibroblasts or cardiomyocytes. Cardiac function was assessed after reperfused or nonreperfused infarction using echocardiography. The effects of cell-specific Smad3 loss on the infarcted heart were studied using histological studies, assessment of protein, and gene expression levels. In vitro, we studied Smad-dependent and Smad-independent actions in isolated cardiac fibroblasts. RESULTS: Mice with fibroblast-specific Smad3 loss had accentuated adverse remodeling after reperfused infarction and exhibited an increased incidence of late rupture after nonreperfused infarction. The consequences of fibroblast-specific Smad3 loss were not a result of effects on acute infarct size but were associated with unrestrained fibroblast proliferation, impaired scar remodeling, reduced fibroblast-derived collagen synthesis, and perturbed alignment of myofibroblast arrays in the infarct. Polarized light microscopy in Sirius red-stained sections demonstrated that the changes in fibroblast morphology were associated with perturbed organization of the collagenous matrix in the infarcted area. In contrast, α-smooth muscle actin expression by infarct myofibroblasts was not affected by Smad3 loss. Smad3 critically regulated fibroblast function, activating integrin-mediated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-2 (NOX-2) expression. Smad3 loss in cardiomyocytes attenuated remodeling and dysfunction after infarction. Cardiomyocyte-specific Smad3 loss did not affect acute infarct size but was associated with attenuated cardiomyocyte apoptosis in the remodeling myocardium, accompanied by decreased myocardial NOX-2 levels, reduced nitrosative stress, and lower matrix metalloproteinase-2 expression. CONCLUSIONS: In healing myocardial infarction, myofibroblast- and cardiomyocyte-specific activation of Smad3 has contrasting functional outcomes that may involve activation of an integrin/reactive oxygen axis.

Immune cells in repair of the infarcted myocardium.

The immune system plays a critical role in both repair and remodeling of the infarcted myocardium. Danger signals released by dying cardiomyocytes mobilize, recruit, and activate immune cells, triggering an inflammatory reaction. CXC chemokines containing the ELR motif attract neutrophils, while CC chemokines mediate recruitment of mononuclear cell subpopulations, contributing to clearance of the infarct from dead cells and matrix debris. Immune cell subsets also participate in suppression and containment of the postinfarction inflammatory response by secreting anti-inflammatory mediators, such as IL-10 and TGF-ß. As proinflammatory signaling is suppressed, macrophage subpopulations, mast cells and lymphocytes, activate fibrogenic and angiogenic responses, contributing to scar formation. In the viable remodeling myocardium, chronic activation of immune cells may promote fibrosis and hypertrophy. This review discusses the role of immune cells in repair and remodeling of the infarcted myocardium. Understanding the role of immune cells in myocardial infarction is critical for the development of therapeutic strategies aimed at protecting the infarcted heart from adverse remodeling. Moreover, modulation of immune cell phenotype may be required in order to achieve the visionary goal of myocardial regeneration.

Effect of damage control strategy combining pre-hospital emergency treatment with in-hospital treatment on pelvic fracture complicated by multiple injuries.

OBJECTIVE: To analyze the application value of damage control strategies combining pre-hospital emergency treatment with in-hospital treatment for multiple injuries in treating pelvic fracture complicated by multiple injuries. METHODS: 120 patients with pelvic fracture complicated by multiple injuries admitted to our hospital from January 2020 to January 2023 were selected and divided into a damage control group (early temporary reduction after resuscitation, n = 60) and a control group (no reduction and resuscitation only, n = 60) by treatment methods. The control group was treated with conventional methods, while the damage control group was treated with the damage control strategy combining pre-hospital emergency treatment combined with in-hospital treatment in addition to conventional methods. The mortality rate, complication rate, fracture reduction quality, long-term efficacy, and patient satisfaction of the two groups were compared. RESULTS: The mortality rate of the damage control group was lower than that of the control group, and the difference has statistical significance (P<0.05); the incidence of infection, DIC, and MODS of the damage control group were lower than that of the control group, with the difference being statistically significant (P<0.05); the incidence of ARDS in the two groups is not that different (P>0.05); the fracture reduction quality and long-term therapeutic effect of patients in the two groups were statistically different, with the damage control group outperforming the control group in both aspects; the difference between the two groups in terms of patient satisfaction was statistically significant (P<0.05), with the patient satisfaction of the damage control group being higher than that of the control group. CONCLUSION: For patients with pelvic fracture, the application of the damage control strategy combining pre-hospital emergency treatment and in-hospital treatment is a boon to the standardization of the treatment process, the improvement of the treatment success rate and fracture reduction quality and the reduction of complications, and therefore is worth promoting in clinical practice. the early application of external fixation has helped with the definitive reduction at a time when the patuent was stable.

Protective effects of macrophage-specific integrin α5 in myocardial infarction are associated with accentuated angiogenesis.

Macrophages sense changes in the extracellular matrix environment through the integrins and play a central role in regulation of the reparative response after myocardial infarction. Here we show that macrophage integrin α5 protects the infarcted heart from adverse remodeling and that the protective actions are associated with acquisition of an angiogenic macrophage phenotype. We demonstrate that myeloid cell- and macrophage-specific integrin α5 knockout mice have accentuated adverse post-infarction remodeling, accompanied by reduced angiogenesis in the infarct and border zone. Single cell RNA-sequencing identifies an angiogenic infarct macrophage population with high Itga5 expression. The angiogenic effects of integrin α5 in macrophages involve upregulation of Vascular Endothelial Growth Factor A. RNA-sequencing of the macrophage transcriptome in vivo and in vitro followed by bioinformatic analysis identifies several intracellular kinases as potential downstream targets of integrin α5. Neutralization assays demonstrate that the angiogenic actions of integrin α5-stimulated macrophages involve activation of Focal Adhesion Kinase and Phosphoinositide 3 Kinase cascades.

Identification of macrophages in normal and injured mouse tissues using reporter lines and antibodies.

Reliable tools for macrophage identification in mouse tissues are critical for studies investigating inflammatory and reparative responses. Transgenic reporter mice and anti-macrophage antibodies have been used as "specific pan-macrophage" markers in many studies; however, organ-specific patterns of expression and non-specific labeling of other cell types, such as fibroblasts, may limit their usefulness. Our study provides a systematic comparison of macrophage labeling patterns in normal and injured mouse tissues, using the CX3CR1 and CSF1R macrophage reporter lines and anti-macrophage antibodies. Moreover, we tested the specificity of macrophage antibodies using the fibroblast-specific PDGFR[Formula: see text] reporter line. Mouse macrophages exhibit organ-specific differences in expression of macrophage markers. Hepatic macrophages are labeled for CSF1R, Mac2 and F4/80, but lack CX3CR1 expression, whereas in the lung, the CSF1R+/Mac2+/Mac3+ macrophage population is not labeled with F4/80. In the splenic red pulp, subpopulations of CSF1R+/F4/80+/Mac3+cells were labeled with Mac2, CX3CR1 and lysozyme M. In the kidney, Mac2, Mac3 and lysozyme M labeled a fraction of the CSF1R+ and CX3CR1+ macrophages, but also stained tubular epithelial cells. In normal hearts, the majority of CSF1R+ and CX3CR1+ cells were not detected with anti-macrophage antibodies. Myocardial infarction was associated with marked expansion of the CSF1R+ and CX3CR1+ populations that peaked during the proliferative phase of cardiac repair, and also expressed Mac2, Mac3 and lysozyme M. In normal mouse tissues, a small fraction of cells labeled with anti-macrophage antibodies were identified as PDGFR[Formula: see text]+ fibroblasts, using a reporter system. The population of PDGFR[Formula: see text]+ cells expressing macrophage markers expanded following injury, likely reflecting emergence of cellular phenotypes with both fibroblast and macrophage characteristics. In conclusion, mouse macrophages exhibit remarkable heterogeneity. Selection of the most appropriate markers for identification of macrophages in mouse tissues is dependent on the organ and the pathologic condition studied.

Smad7 effects on TGF-ß and ErbB2 restrain myofibroblast activation and protect from postinfarction heart failure.

Repair of the infarcted heart requires TGF-ß/Smad3 signaling in cardiac myofibroblasts. However, TGF-ß-driven myofibroblast activation needs to be tightly regulated in order to prevent excessive fibrosis and adverse remodeling that may precipitate heart failure. We hypothesized that induction of the inhibitory Smad, Smad7, may restrain infarct myofibroblast activation, and we examined the molecular mechanisms of Smad7 actions. In a mouse model of nonreperfused infarction, Smad3 activation triggered Smad7 synthesis in α-SMA+ infarct myofibroblasts, but not in α-SMA-PDGFRα+ fibroblasts. Myofibroblast-specific Smad7 loss increased heart failure-related mortality, worsened dysfunction, and accentuated fibrosis in the infarct border zone and in the papillary muscles. Smad7 attenuated myofibroblast activation and reduced synthesis of structural and matricellular extracellular matrix proteins. Smad7 effects on TGF-ß cascades involved deactivation of Smad2/3 and non-Smad pathways, without any effects on TGF-ß receptor activity. Unbiased transcriptomic and proteomic analysis identified receptor tyrosine kinase signaling as a major target of Smad7. Smad7 interacted with ErbB2 in a TGF-ß-independent manner and restrained ErbB1/ErbB2 activation, suppressing fibroblast expression of fibrogenic proteases, integrins, and CD44. Smad7 induction in myofibroblasts serves as an endogenous TGF-ß-induced negative feedback mechanism that inhibits postinfarction fibrosis by restraining Smad-dependent and Smad-independent TGF-ß responses, and by suppressing TGF-ß-independent fibrogenic actions of ErbB2.

Chemokines in Myocardial Infarction.

In the infarcted myocardium, cardiomyocyte necrosis triggers an intense inflammatory reaction that not only is critical for cardiac repair, but also contributes to adverse remodeling and to the pathogenesis of heart failure. Both CC and CXC chemokines are markedly induced in the infarcted heart, bind to endothelial glycosaminoglycans, and regulate leukocyte trafficking and function. ELR+ CXC chemokines (such as CXCL8) control neutrophil infiltration, whereas CC chemokines (such as CCL2) mediate recruitment of mononuclear cells. Moreover, some members of the chemokine family (such as CXCL10 and CXCL12) may mediate leukocyte-independent actions, directly modulating fibroblast and vascular cell function. This review manuscript discusses our understanding of the role of the chemokines in regulation of injury, repair, and remodeling following myocardial infarction. Although several chemokines may be promising therapeutic targets in patients with myocardial infarction, clinical implementation of chemokine-based therapeutics is hampered by the broad effects of the chemokines in both injury and repair.

Activation of Pancreatic Stellate Cells Is Beneficial for Exocrine but Not Endocrine Cell Differentiation in the Developing Human Pancreas.

Pancreatic stellate cells (PaSCs) are non-endocrine, mesenchymal-like cells that reside within the peri-pancreatic tissue of the rodent and human pancreas. PaSCs regulate extracellular matrix (ECM) turnover in maintaining the integrity of pancreatic tissue architecture. Although there is evidence indicating that PaSCs are involved in islet cell survival and function, its role in islet cell differentiation during human pancreatic development remains unclear. The present study examines the expression pattern and functional role of PaSCs in islet cell differentiation of the developing human pancreas from late 1st to 2nd trimester of pregnancy. The presence of PaSCs in human pancreata (8-22 weeks of fetal age) was characterized by ultrastructural, immunohistological, quantitative RT-PCR and western blotting approaches. Using human fetal PaSCs derived from pancreata at 14-16 weeks, freshly isolated human fetal islet-epithelial cell clusters (hIECCs) were co-cultured with active or inactive PaSCs in vitro. Ultrastructural and immunofluorescence analysis demonstrated a population of PaSCs near ducts and newly formed islets that appeared to make complex cell-cell dendritic-like contacts. A small subset of PaSCs co-localized with pancreatic progenitor-associated transcription factors (PDX1, SOX9, and NKX6-1). PaSCs were highly proliferative, with significantly higher mRNA and protein levels of PaSC markers (desmin, αSMA) during the 1st trimester of pregnancy compared to the 2nd trimester. Isolated human fetal PaSCs were identified by expression of stellate cell markers and ECM. Suppression of PaSC activation, using all-trans retinoic acid (ATRA), resulted in reduced PaSC proliferation and ECM proteins. Co-culture of hIECCs, directly on PaSCs or indirectly using Millicell® Inserts or using PaSC-conditioned medium, resulted in a reduction the number of insulin+ cells but a significant increase in the number of amylase+ cells. Suppression of PaSC activation or Notch activity during the co-culture resulted in an increase in beta-cell differentiation. This study determined that PaSCs, abundant during the 1st trimester of pancreatic development but decreased in the 2nd trimester, are located near ductal and islet structures. Direct and indirect co-cultures of hIECCs with PaSCs suggest that activation of PaSCs has opposing effects on beta-cell and exocrine cell differentiation during human fetal pancreas development, and that these effects may be dependent on Notch signaling.

The role of Smad2 and Smad3 in regulating homeostatic functions of fibroblasts in vitro and in adult mice.

The heart contains an abundant fibroblast population that may play a role in homeostasis, by maintaining the extracellular matrix (ECM) network, by regulating electrical impulse conduction, and by supporting survival and function of cardiomyocytes and vascular cells. Despite an explosion in our understanding of the role of fibroblasts in cardiac injury, the homeostatic functions of resident fibroblasts in adult hearts remain understudied. TGF-ß-mediated signaling through the receptor-activated Smads, Smad2 and Smad3 critically regulates fibroblast function. We hypothesized that baseline expression of Smad2/3 in fibroblasts may play an important role in cardiac homeostasis. Smad2 and Smad3 were constitutively expressed in normal mouse hearts and in cardiac fibroblasts. In cultured cardiac fibroblasts, Smad2 and Smad3 played distinct roles in regulation of baseline ECM gene synthesis. Smad3 knockdown attenuated collagen I, collagen IV and fibronectin mRNA synthesis and reduced expression of the matricellular protein thrombospondin-1. Smad2 knockdown on the other hand attenuated expression of collagen V mRNA and reduced synthesis of fibronectin, periostin and versican. In vivo, inducible fibroblast-specific Smad2 knockout mice and fibroblast-specific Smad3 knockout mice had normal heart rate, preserved cardiac geometry, ventricular systolic and diastolic function, and normal myocardial structure. Fibroblast-specific Smad3, but not Smad2 loss modestly but significantly reduced collagen content. Our findings suggest that fibroblast-specific Smad3, but not Smad2, may play a role in regulation of baseline collagen synthesis in adult hearts. However, at least short term, these changes do not have any impact on homeostatic cardiac function.

Islet Stellate Cells Isolated from Fibrotic Islet of Goto-Kakizaki Rats Affect Biological Behavior of Beta-Cell.

We previously isolated islet stellate cells (ISCs) from healthy Wistar rat islets. In the present study, we isolated "already primed by diabetic environment" ISCs from islets of Goto-Kakizaki rats, determined the gene profile of these cells, and assessed the effects of these ISCs on beta-cell function and survival. We detected gene expression of ISCs by digital gene expression. INS-1 cell proliferation, apoptosis, and insulin production were measured after being treated with ISCs supernatant (SN). We observed the similar expression pattern of ISCs and PSCs, but 1067 differentially expressed genes. Insulin production in INS-1 cells cultured with ISC-SN was significantly reduced. The 5-ethynyl-2'-deoxyuridine-positive INS-1 cells treated with ISC-SN were decreased. Propidium iodide- (PI-) positive INS-1 cells were 2.6-fold higher than those in control groups. Caspase-3 activity was increased. In conclusion, ISCs presented in fibrotic islet of GK rats might be special PSCs, which impaired beta-cell function and proliferation and increased beta-cell apoptosis.

Maintaining human fetal pancreatic stellate cell function and proliferation require ß1 integrin and collagen I matrix interactions.

Pancreatic stellate cells (PaSCs) are cells that are located around the acinar, ductal, and vasculature tissue of the rodent and human pancreas, and are responsible for regulating extracellular matrix (ECM) turnover and maintaining the architecture of pancreatic tissue. This study examines the contributions of integrin receptor signaling in human PaSC function and survival. Human PaSCs were isolated from pancreata collected during the 2nd trimester of pregnancy and identified by expression of stellate cell markers, ECM proteins and associated growth factors. Multiple integrins are found in isolated human PaSCs, with high levels of ß1, α3 and α5. Cell adhesion and migration assays demonstrated that human PaSCs favour collagen I matrix, which enhanced PaSC proliferation and increased TGFß1, CTGF and α3ß1 integrin. Significant activation of FAK/ERK and AKT signaling pathways, and up-regulation of cyclin D1 protein levels, were observed within PaSCs cultured on collagen I matrix. Blocking ß1 integrin significantly decreased PaSC adhesion, migration and proliferation, further complementing the aforementioned findings. This study demonstrates that interaction of ß1 integrin with collagen I is required for the proliferation and function of human fetal PaSCs, which may contribute to the biomedical engineering of the ECM microenvironment needed for the efficient regulation of pancreatic development.

Pancreatic stone protein/regenerating protein (PSP/reg): a novel secreted protein up-regulated in type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) has insulin resistance (IR) or reduced ß-cell mass, partially due to an increased ß-cell apoptosis rate. Pancreatic stone protein/regenerating protein (PSP/reg) is a secretory protein produced in the pancreas and up-regulated dramatically during pancreatic disease. Recent studies revealed that ß-cells undergoing apoptosis induce PSP/reg expression in surviving neighboring cells. Further experiments demonstrated that PSP/reg was elevated during disease progression in type 1 diabetes mellitus (T1DM). However, the association between PSP/reg and T2DM patients is unknown. The aim of this pilot study was to investigate PSP/reg in different clinical stages of T2DM and evaluate its correlation with chronic complications of diabetes. A total of 1,121 participants (479 males, 642 females; age range 23-80 years) were enrolled in this study. PSP/reg serum values were measured by a newly developed enzyme-linked immunosorbent assay (ELISA). We analyzed its correlation with clinical and biochemical parameters in subjects with T2DM at different clinical phases. Statistical analyses were conducted using SPSS 17.0 software. Correlations of PSP/reg and clinical parameters were performed using Spearman's rank correlation coefficient. Differences between groups were determined by Nemenyi test. PSP/reg was elevated in high-risk and impaired glucose regulation (IGR) patients (p<0.05). PSP/reg was significantly up-regulated in newly diagnosed T2DM patients and long-term diabetes patients with complications (p<0.001). PSP/reg levels correlated with the duration of diabetes (p<0.001). The area under the curve (AUC) for presence of diabetes-onset and its chronic complications was 0.640 and 0.754, respectively. PSP/reg is significantly up-regulated in T2DM patients, and PSP/reg levels are related to the duration of diabetes. Therefore, PSP/reg might be useful as a predictor of T2DM and disease progression.