Wenli Yang

The Role of CX3CL1/CX3CR1 in Pulmonary Angiogenesis and Intravascular Monocyte Accumulation in Rat Experimental Hepatopulmonary Syndrome

BACKGROUND & AIMS Hepatopulmonary syndrome (HPS), classically attributed to intrapulmonary vascular dilatation, occurs in 15-30% of cirrhotics and causes hypoxemia and increases mortality. In experimental HPS after common bile duct ligation (CBDL), monocytes adhere in the lung vasculature and produce vascular endothelial growth factor (VEGF)-A and angiogenesis ensues and contribute to abnormal gas exchange. However, the mechanisms for these events are unknown. The chemokine fractalkine (CX(3)CL1) can directly mediate monocyte adhesion and activate VEGF-A and angiogenesis via its receptor CX(3)CR1 on monocytes and endothelium during inflammatory angiogenesis. We explored whether pulmonary CX(3)CL1/CX(3)CR1 alterations occur after CBDL and influence pulmonary angiogenesis and HPS. METHODS Pulmonary CX(3)CL1/CX(3)CR1 expression and localization, CX(3)CL1 signaling pathway activation, monocyte accumulation, and development of angiogenesis and HPS were assessed in 2- and 4-week CBDL animals. The effects of a neutralizing antibody to CX(3)CR1 (anti-CX(3)CR1 Ab) on HPS after CBDL were evaluated. RESULTS Circulating CX(3)CL1 levels and lung expression of CX(3)CL1 and CX(3)CR1 in intravascular monocytes and microvascular endothelium increased in 2- and 4-week CBDL animals as HPS developed. These events were accompanied by pulmonary angiogenesis, monocyte accumulation, activation of CX(3)CL1 mediated signaling pathways (Akt, ERK) and increased VEGF-A expression and signaling. Anti-CX(3)CR1 Ab treatment reduced monocyte accumulation, decreased lung angiogenesis and improved HPS. These events were accompanied by inhibition of CX(3)CL1 signaling pathways and a reduction in VEGF-A expression and signaling. CONCLUSIONS Circulating CX(3)CL1 levels and pulmonary CX(3)CL1/CX(3)CR1 expression and signaling increase after CBDL and contribute to pulmonary intravascular monocyte accumulation, angiogenesis and development of experimental HPS.

BMP2 inhibits TGF-β-induced pancreatic stellate cell activation and extracellular matrix formation

Activation of pancreatic stellate cells (PSCs) by transforming growth factor (TGF)-β is the key step in the development of pancreatic fibrosis, a common pathological feature of chronic pancreatitis (CP). Bone morphogenetic proteins (BMPs), members of the TGF-β superfamily, have anti-fibrogenic functions, in contrast to TGF-β, in the kidney, lung, and liver. However, it is not known whether BMPs have an anti-fibrogenic role in the pancreas. The current study was designed to investigate the potential anti-fibrogenic role of BMPs in the pancreas using an in vivo CP model and an in vitro PSC model. CP was induced by repetitive intraperitoneal injections of cerulein in adult Swiss Webster mice. The control mice received saline injections. Compared with the control, cerulein injections induced a time-dependent increase in acinar injury and progression of fibrosis and a steady increase in inflammation. Cerulein injections also induced increases of the extracellular matrix (ECM) protein fibronectin and of α-smooth muscle actin (α-SMA)-positive stellate cells (PSCs). The mice receiving cerulein injections showed increased BMP2 protein levels and phosphorylated Smad1 levels up to 4 wk and then declined at 8 wk to similar levels as the control. In vitro, the isolated mouse and human PSCs were cultured and pretreated with BMP2 followed by TGF-β treatment. BMP2 pretreatment inhibited TGF-β-induced α-SMA, fibronectin, and collagen type Ia expression. Knocking down Smad1 with small-interfering RNA reversed the inhibitory effect of BMP2 on TGF-β-induced α-SMA and fibronectin expression. Thus, BMP2 opposes the fibrogenic function of TGF-β in PSCs through the Smad1 signaling pathway.

Endothelin-1 activation of the endothelin B receptor modulates pulmonary endothelial CX3CL1 and contributes to pulmonary angiogenesis in experimental hepatopulmonary syndrome.

Hepatic production and release of endothelin-1 (ET-1) binding to endothelin B (ETB) receptors, overexpressed in the lung microvasculature, is associated with accumulation of pro-angiogenic monocytes and vascular remodeling in experimental hepatopulmonary syndrome (HPS) after common bile duct ligation (CBDL). We have recently found that lung vascular monocyte adhesion and angiogenesis in HPS involve interaction of endothelial C-X3-C motif ligand 1 (CX3CL1) with monocyte CX3C chemokine receptor 1 (CX3CR1), although whether ET-1/ETB receptor activation influences these events is unknown. Our aim was to define if ET-1/ETB receptor activation modulates CX3CL1/CX3CR1 signaling and lung angiogenesis in experimental HPS. A selective ETB receptor antagonist, BQ788, was given for 2 weeks to 1-week CBDL rats. ET-1 (±BQ788) was given to cultured rat pulmonary microvascular endothelial cells overexpressing ETB receptors. BQ788 treatment significantly decreased lung angiogenesis, monocyte accumulation, and CX3CL1 levels after CBDL. ET-1 treatment significantly induced CX3CL1 production in lung microvascular endothelial cells, which was blocked by inhibitors of Ca(2+) and mitogen-activated protein kinase (MEK)/ERK pathways. ET-1-induced ERK activation was Ca(2+) independent. ET-1 administration also increased endothelial tube formation in vitro, which was inhibited by BQ788 or by blocking Ca(2+) and MEK/ERK activation. CX3CR1 neutralizing antibody partially inhibited ET-1 effects on tube formation. These findings identify a novel mechanistic interaction between the ET-1/ETB receptor axis and CX3CL1/CX3CR1 in mediating pulmonary angiogenesis and vascular monocyte accumulation in experimental HPS.

Penile rehabilitation with a vacuum erectile device in an animal model is related to an antihypoxic mechanism： blood gas evidence

Our previous study showed that vacuum erectile device (VED) therapy has improved erectile function in rats with bilateral cavernous nerve crush (BCNC) injuries. This study was designed to explore the mechanism of VED in penile rehabilitation by analyzing cavernous oxygen saturation (SO2) and to examine the effect of VED therapy on preventing penile shrinkage after BCNC. Thirty adult Sprague-Dawley rats were randomly assigned into three groups: group 1, sham surgery; group 2, BCNC; and group 3, BCNC+VED. Penile length and diameter were measured on a weekly basis. After 4 weeks of therapy, the penile blood was extracted by three methods for blood gas analysis (BGA): method 1, cavernous blood was aspirated at the flaccid state; method 2, cavernous blood was aspirated at the traction state; and method 3, cavernous blood was aspirated immediately after applying VED. SO2 values were tested by the blood gas analyzer. The results showed that VED therapy is effective in preventing penile shrinkage induced by BCNC (Penile shortening: BCNC group 1.9±1.1 mm; VED group 0.3±1.0 mm; P<0.01. Penile diameter reduction: BCNC group 0.28±0.14 mm; VED group 0.04±0.14 mm; P<0.01). The mean SO2±s.d. values were increased by VED application (88.25%±4.94%) compared to the flaccid (76.53%±4.16%) or traction groups (78.93%±2.56%) (P<0.05). The calculated blood constructs in the corpus cavernosum right after VED application were 62% arterial and 38% venous blood. These findings suggest that VED therapy can effectively preserve penile size in rats with BCNC injury. The beneficial effect of VED therapy is related to antihypoxia by increasing cavernous blood SO2.

The role of receptor tyrosine kinase activation in cholangiocytes and pulmonary vascular endothelium in experimental hepatopulmonary syndrome

Pulmonary vascular dilation and angiogenesis underlie experimental hepatopulmonary syndrome (HPS) induced by common bile duct ligation (CBDL) and may respond to receptor tyrosine kinase (RTK) inhibition. Vascular endothelial growth factor-A (VEGF-A) expression occurs in proliferating cholangiocytes and pulmonary intravascular monocytes after CBDL, the latter contributing to angiogenesis. CBDL cholangiocytes also produce endothelin-1 (ET-1), which triggers lung vascular endothelin B receptor-mediated endothelial nitric oxide synthase (eNOS) activation and pulmonary intravascular monocyte accumulation. However, whether RTK pathway activation directly regulates cholangiocyte and pulmonary microvascular alterations in experimental HPS is not defined. We assessed RTK pathway activation in cholangiocytes and lung after CBDL and the effects of the type II RTK inhibitor sorafenib in experimental HPS. Cholangiocyte VEGF-A expression and ERK activation accompanied proliferation and increased hepatic and circulating ET-1 levels after CBDL. Sorafenib decreased each of these events and led to a reduction in lung eNOS activation and intravascular monocyte accumulation. Lung monocyte VEGF-A expression and microvascular Akt and ERK activation were also found in vivo after CBDL, and VEGF-A activated Akt and ERK and angiogenesis in rat pulmonary microvascular endothelial cells in vitro. Sorafenib inhibited VEGF-A-mediated signaling and angiogenesis in vivo and in vitro and improved arterial gas exchange and intrapulmonary shunting. RTK activation in experimental HPS upregulates cholangiocyte proliferation and ET-1 production, leading to pulmonary microvascular eNOS activation, intravascular monocyte accumulation, and VEGF-A-mediated angiogenic signaling pathways. These findings identify a novel mechanism in cholangiocytes through which RTK inhibition ameliorates experimental HPS.

COX-2-10aa-PGIS Gene Therapy Improves Erectile Function in Rats after Cavernous Nerve Injury

INTRODUCTION Erectile dysfunction (ED) is a very common complication after radical prostatectomy. COX-2-10aa-PGIS is a newly engineered protein with COX-2 and prostacyclin synthase activities that converts arachidonic acid directly to prostacyclin (prostaglandin I2 [PGI2]). PGI2 is a potent smooth muscle relaxant. AIM The purpose of this study was to explore the effect and mechanism of COX-2-10aa-PGIS gene therapy in penile rehabilitation. METHODS Bilateral cavernous nerve crush (BCNC) in adult Sprague-Dawley rats was used to mimic radical prostatectomy-induced ED. Sprague-Dawley rats were randomly assigned into four groups: 1. sham surgery; 2. BCNC; 3. BCNC + null control recombinant adenovirus intracavernous injection; and 4. BCNC + Ad-COX2-10aa-PGIS intracavernous injection. Twenty-eight days later, intracavernosal pressure (ICP) was recorded under cavernous nerve stimulation; in the meantime, the mean arterial pressure (MAP) was monitored. At the end of the measurement, the penis was harvested and processed for (i) immunohistochemistry analysis of endothelial nitric oxide synthase (eNOS), alpha-smooth muscle actin (α-SMA), and transforming growth factor beta-1 (TGF-β1); (ii) Massons trichrome stain for smooth muscle/collagen ratios; (iii) Western blot of eNOS, α-SMA, TGF-β1, and COX2-10aa-PGIS; and (iv) terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for apoptosis. MAIN OUTCOME MEASURES Erectile function was evaluated by ICP/MAP. Smooth muscle and endothelium functions in corpora cavernosum were assessed by Massons trichrome stain, immunohistochemistry, and Western blot. Apoptosis was identified by TUNEL assay. RESULTS The results were the following: 1. COX2-10aa-PGIS gene therapy improved erectile function (82%, compared with control) in the BCNC rat model; 2. COX2-10aa-PGIS gene therapy increased eNOS (121%) and α-SMA (118%) expression and decreased TGF-β1 (45%) expression; 3. COX2-10aa-PGIS gene therapy reduced cell apoptosis after cavernous nerve injury (64%); and 4. COX2-10aa-PGIS gene therapy improved smooth muscle/collagen ratios (81%). CONCLUSION Our data demonstrated that COX2-10aa-PGIS improved erectile function after cavernous nerve injury through antifibrotic and anti-apoptotic mechanisms.

Noggin attenuates cerulein-induced acute pancreatitis and impaired autophagy

Objectives To investigate the role of bone morphogenetic protein (BMP) signaling in acute pancreatitis (AP) by administration of noggin, an endogenous BMP antagonist, in a cerulein-induced AP model. Methods Acute pancreatitis was induced by 9 hourly intraperitoneal injections of cerulein (50 &mgr;g/kg). Control mice received phosphate-buffered saline injections. In a separate group, noggin (0.5 mg/kg) was given intraperitoneally at 1 hour before and 2, 4, and 6 hours after AP induction. The mice were euthanized at 1 hour after completion of AP induction. The blood samples and the pancreas were harvested for analysis. Isolated pancreatic acini from normal mice and AR42J cells were treated with BMP2 and cerulein. AR42J cells were also treated with noggin. Phosphorylation of Smad1/5/8 was measured. Results Bone morphogenetic protein signaling was up-regulated in AP mouse pancreas. Bone morphogenetic protein 2 and cerulein-induced phosphorylation of Smad1/5/8 in the acinar cells in vitro, which was blocked by noggin. Noggin administration in vivo attenuated AP induction, decreased vacuole formation in acinar cells, blocked LC3-II levels, and partially restored Beclin-1 and lysosomal-associated membrane protein 2 levels. Conclusions Bone morphogenetic protein signaling seems to promote AP induction and autophagy, as suggested by our study showing that noggin ameliorates AP and partially restores autophagic homeostasis.

Alveolar Type II Epithelial Cell Dysfunction in Rat Experimental Hepatopulmonary Syndrome (HPS)

The hepatopulmonary syndrome (HPS) develops when pulmonary vasodilatation leads to abnormal gas exchange. However, in human HPS, restrictive ventilatory defects are also observed supporting that the alveolar epithelial compartment may also be affected. Alveolar type II epithelial cells (AT2) play a critical role in maintaining the alveolar compartment by producing four surfactant proteins (SPs, SP-A, SP-B, SP-C and SP-D) which also facilitate alveolar repair following injury. However, no studies have evaluated the alveolar epithelial compartment in experimental HPS. In this study, we evaluated the alveolar epithelial compartment and particularly AT2 cells in experimental HPS induced by common bile duct ligation (CBDL). We found a significant reduction in pulmonary SP production associated with increased apoptosis in AT2 cells after CBDL relative to controls. Lung morphology showed decreased mean alveolar chord length and lung volumes in CBDL animals that were not seen in control models supporting a selective reduction of alveolar airspace. Furthermore, we found that administration of TNF-α, the bile acid, chenodeoxycholic acid, and FXR nuclear receptor activation (GW4064) induced apoptosis and impaired SP-B and SP-C production in alveolar epithelial cells in vitro. These results imply that AT2 cell dysfunction occurs in experimental HPS and is associated with alterations in the alveolar epithelial compartment. Our findings support a novel contributing mechanism in experimental HPS that may be relevant to humans and a potential therapeutic target.

Increased transforming growth factor β contributes to deterioration of refrigerated fresh frozen plasma's effects in vitro on endothelial cells

Resuscitation with fresh frozen plasma (FFP) is associated with improved outcomes after hemorrhagic shock. Many trauma centers are using thawed plasma that has been stored for up to 5 days at 4°C (refrigeration), yet the effect of refrigeration on FFP is relatively unknown. Previously, our group showed that refrigeration of FFP changed its coagulation factors and diminished its beneficial effects on endothelial cell (EC) function and resuscitation in an animal model of hemorrhagic shock. We hypothesize that growth factor composition of FFP is altered during refrigeration, leading to a diminished beneficial effect on EC. Transforming growth factor (TGF-&bgr;) is a potent inhibitor of EC migration and is released during refrigeration of platelets. We found increased TGF-&bgr;1 protein levels and greater activation of downstream mediators Smad2/3 during refrigeration of FFP. Both day 0 FFP (used on the same day after being thawed) and day 5 FFP (used after being thawed and refrigerated for 5 days) stimulated EC migration in vitro; however, the EC migration in day 5 FFP was significantly reduced. Inhibition of TGF-&bgr; type I receptor blocked FFP-induced Smad3 signaling in EC cells and restored the effectiveness of day 5 FFP on EC migration to a comparable level seen in day 0 FFP. These data suggest that the increased TGF-&bgr; levels during FFP refrigeration contribute to the deterioration of refrigerated FFPs effects on EC migration. This study identifies a novel molecular mechanism contributing to the reduced efficacy of refrigerated FFP.

Role of splenic reservoir monocytes in pulmonary vascular monocyte accumulation in experimental hepatopulmonary syndrome

Pulmonary monocyte infiltration plays a significant role in the development of angiogenesis in experimental hepatopulmonary syndrome (HPS) after common bile duct ligation (CBDL). Hepatic monocytes are also increased after CBDL, but the origins remain unclear. Splenic reservoir monocytes have been identified as a major source of monocytes that accumulate in injured tissues. Whether splenic monocytes contribute to monocyte alterations after CBDL is unknown. This study evaluates monocyte distributions and assesses effects of splenectomy on monocyte levels and pulmonary vascular and hepatic abnormalities in experimental HPS.