Successful treatment of stage IIIB intrahepatic cholangiocarcinoma using neoadjuvant therapy with the PD-1 inhibitor camrelizumab: A case report.

BACKGROUND: The prognosis of intrahepatic cholangiocarcinoma (ICC) with lymph node metastasis is poor. The feasibility of surgery is not certain, which is a contraindication according to the National Comprehensive Cancer Network guidelines. The role of immunotherapy as a neoadjuvant therapy for ICC is not clear. We herein describe a case of ICC with lymph node metastasis that was successfully treated with neoadjuvant therapy. CASE SUMMARY: A 60-year-old man with a liver tumor was admitted to our hospital. Enhanced computed tomography and magnetic resonance imaging revealed a space-occupying lesion in the right lobe of the liver. Multiple subfoci were found around the tumor, and the right posterior branch of the portal vein was invaded. Liver biopsy indicated poorly differentiated cholangiocytes. According to the American Joint Committee on Cancer disease stage classification, ICC with hilar lymph node metastasis (stage IIIB) and para-aortic lymph node metastasis was suspected. A report showed that two patients with stage IIIB ICC achieved a complete response (CR) 13 mo and 16 mo after chemotherapy with a PD-1 monoclonal antibody. After multidisciplinary consultation, the patient was given neoadjuvant therapy, surgical resection and lymph node dissection, and postoperative adjuvant therapy. After three rounds of PD-1 immunotherapy (camrelizumab) and two rounds of gemcitabine combined with cisplatin regimen chemotherapy, the tumor size was reduced. Therefore, a partial response was achieved. Exploratory laparotomy found that the lymph nodes of Group 16 were negative, and the tumor could be surgically removed. Therefore, the patient underwent right hemihepatectomy plus lymph node dissection. The patient received six rounds of chemotherapy and five rounds of PD-1 treatment postoperatively. After 8 mo of follow-up, no recurrence was found, and a CR was achieved. CONCLUSION: Neoadjuvant therapy combined with surgical resection is useful for advanced-stage ICC. This is the first report of successful treatment of stage IIIB ICC using neoadjuvant therapy with a PD-1 inhibitor.

LncRNA CRNDE is involved in radiation resistance in hepatocellular carcinoma via modulating the SP1/PDK1 axis.

Hepatocellular carcinoma (HCC) is defined as a universal malignancy while radiation therapy is the effective treatment for it. This study validated the mechanism of long non-coding RNA (lncRNA) colorectal neoplasia differentially expressed gene (CRNDE) in radiation resistance in HCC. LncRNA CRNDE upregulation was detected in HCC cells. The radiation-resistant cell strains Huh7R and SNU-387R were established. After silencing lncRNA CRNDE, the cell colony formation ability, cell activity, apoptosis, cell cycles, and γ-H2AX positive rate in Huh7R and SNU-387R were detected. Silencing lncRNA CRNDE decreased the cell activity, colony formation ability, and cell number in the G2 phase and facilitated DNA damage and apoptosis. The binding relations of specificity protein 1 (SP1) with lncRNA CRNDE and 3-phosphoinositide dependent protein kinase 1 (PDK1) were verified. LncRNA CRNDE regulated PDK1 transcription by binding to transcription factor SP1. PDK1 overexpression partially reversed the inhibition of silencing lncRNA CRNDE on radiation resistance in HCC cells. The transplanted tumor mouse model was established and showed that silencing lncRNA CRNDE decreased tumor volume and weight and Ki67-positive cells in HCC mice in vivo. Collectively, lncRNA CRNDE was upregulated in HCC cells and promoted PDK1 transcription by binding to SP1, thus enhancing radiation resistance in HCC cells.

Culture and purification of SD rat corpus cavernosum endothelial cells by enzymatic digestion combined with mechanical extrusion and fixed-point digestion.

To explore a new method of in vitro culture and purification of rat corpus cavernosum endothelial cells (CCECs). Male Sprague-Dawley rats' penile tissue were digested with elastase or collagenase combined with mechanical extrusion to isolate and culture the CCECs. The fixed-point digestion method was used to purify the primary cells. High-purity CCECs were successfully isolated. Following the digestion of the primary CCECs by elastase or collagenase coupled with mechanical extrusion, the cells were paving stone- and cobblestone-shaped over 10 days. The cell purity yielded in the second generation (P2) CCECs after using the fixed-point digestion method was significantly high. Compared with primary CCECs extracted by elastase digestion combined with the mechanical extrusion method, CCECs cultured by collagenase digestion yielded higher purity and a more stable morphology after fixed-point digestion and purification. Immunofluorescence staining of the third generation CCECs and the expression results of endothelial cell-associated marker antibodies CD31 and VWF were positive, and flow cytometry showed the purity of CCECs was 96.9%. Enzymatic digestion combined with mechanical extrusion and fixed-point digestion is a simple, economical method for in vitro culture and purification of CCECs, which is conducive to studying the pathophysiological mechanisms of endothelial dysfunction and erectile dysfunction.

Myxomatous liposarcoma in giant pleural cavity: case report and literature review.

Liposarcoma was mainly occurring in the lower extremities and deep retroperitoneal soft tissues, but rarely occurred in the thoracic cavity. Most cases were reported in the literature, and most of them were middle-aged and elderly people. It was even more rare in adolescents with tumors occupying the entire thoracic cavity. Recently, one case of myxomatous liposarcoma was admitted and treated in our hospital. This paper reports this case and discusses the diagnostic method, pathological type and treatment of giant liposarcoma in the chest, in order to provide a reference for the clinical diagnosis and treatment of this rare tumor.

Achievement of complete response to nivolumab in a patient with advanced sarcomatoid hepatocellular carcinoma: A case report.

BACKGROUND: Sarcomatoid hepatocellular carcinoma (SHC) is a rare subtype of hepatocellular carcinoma (HCC), with a high recurrence rate after surgery. In addition to limited effective treatment for the advanced stage of SHC, the prognosis of patients with this malignancy is worse than that of patients with conventional HCC. CASE SUMMARY: We present the case of a 54-year-old man with SHC who underwent radical segmental hepatectomy, which relapsed 4 mo after surgery due to lymphatic metastasis in the porta hepatis. Although a second surgery was performed, new metastasis developed in the mediastinal lymph nodes. Therefore, sorafenib and lenvatinib were sequentially administered as first- and second-line systemic therapies, respectively. However, progressive disease was confirmed based on a recurrent hepatic lesion and new metastatic lesion in the abdominal cavity. Percutaneous transhepatic cholangial drainage was performed to alleviate the biliary obstruction. Because the tumor was strongly positive for programmed death-ligand 1, the patient was started on nivolumab. Imaging studies revealed that after two cycles of immunotherapy, the metastatic lesions decreased to undetectable levels. CONCLUSION: The patient experienced continuous complete remission for 8 mo. Immune checkpoint inhibitors are useful for the treatment of advanced SHC.

Effect of inorganic carbon limitation on the conversion of organic carbon to total fatty acids by Monodus subterraneus.

Traditional autotrophic microalgae exhibit low rates of organic carbon assimilation and conversion to useful compounds when switching to mixotrophic or heterotrophic growth. The goal of this study was to investigate the effect of inorganic carbon limitation on the efficiency of organic carbon (glycerol) assimilation and conversion to total fatty acids (TFAs) or the long-chain polyunsaturated fatty acid eicosapentaenoic acid (EPA). An oleaginous Monodus subterraneus was selected and six cultivation conditions were set, including Autotrophy-no aeration, Autotrophy-aeration, Mixotrophy-no aeration, Mixotrophy-no aeration & no Na2CO3, Mixotrophy-aeration, and Heterotrophy. The results showed M. subterraneus could utilize glycerol and grow under mixotrophic condition, while it was not occurred under heterotrophy. Superiority of mixotrophy to autotrophy on biomass productivity was more obvious under inorganic carbon limitation (no aeration or no Na2CO3) than inorganic carbon supply (aeration and existing Na2CO3 in the medium). CO2 limitation (no aeration) decreased content (of dry weight) and production (in medium) of TFAs, which was not evident in mixotrophy. CO2 limitation and inorganic carbon substrate stress largely improved the COD yield of TFAs and EPA under mixotrophic condition. TFA yield (%COD) under Mixotrophy-no aeration & no Na2CO3 was maximum (22.82%), and was almost two-fold higher than that under Mixotrophy-no aeration and nearly three-fold higher than that with Mixotrophy-aeration. EPA yield (% COD) under mixotrophy-no aeration & no Na2CO3 was maximum (6.58%). These results suggested that inorganic carbon limitation is a potentially useful method to enhance conversion of organic carbon to TFAs. Furthermore, the results suggest an application to obtain high value compounds (TFAs or EPA) combined with a high assimilation rate of waste glycerol from biodiesel and epichlorohydrin production by microalgae.

Significant effects of two pesticides on the bacteriostatic activity and antioxidant ability of green tea polyphenols.

Green tea polyphenols (GTPs) are widely used in food preservation because of their strong bacteriostatic activity and antioxidant ability, and whether pesticides as common pollutants in food will affect the function of GTPs is worthy of attention. Therefore, GTPs and two pesticides, namely, acetamiprid (ACE) and diquat dibromide (DIQ) commonly used on food crops were selected as research objects and Vibrio qinghaiensis sp.-Q67 (Q67) as the test organism to explore the effects of pesticide pollutants on the bacteriostatic activity of GTPs by the time-dependent microplate toxicity analysis method (t-MTA). The binary mixture systems of GTPs and two pesticides were designed by the direct equipartition ray design method (EquRay). The effect residual ratio (ERR) method was used to quantify the toxicity interactions of binary mixture systems. Besides, the effects of these two pesticides on the antioxidant capacity of GTPs were investigated. The results indicated that the bacteriostatic activity of GTPs upon interaction with the two pesticides shows certain time characteristics. These two pesticides can affect the bacteriostatic activity of GTPs, which is enhanced or weakened with prolonged duration, i.e. time-dependent synergism or antagonism. The bacteriostatic mechanism of the three substances and their mixtures is produced by affecting the cell morphology or destroying the cell structure, and the long-term antagonism of the three substances may be due to the competition for the active site. The two pesticides can greatly reduce the antioxidant capacity of GTPs. ACE reduces the free radical scavenging ability of GTPs by 14-24% and DIQ by 39-63% in the experimental concentration ratios. In addition, the free radical scavenging ability of GTPs decreases with the increase in the concentration ratio of the two pesticides in the mixture systems.

Preliminary study of magnetically controlled capsule gastroscopy for diagnosing superficial gastric neoplasia.

BACKGROUND: Magnetically controlled capsule gastroscopy (MCCG) is a newly developed non-invasive method designed for gastric examination. Although favorable diagnostic accuracy has been reported, there is little if any data about its ability to diagnose gastric cancer. AIMS: To compare the detectability of superficial gastric neoplasia by MCCG and gastroscopy. METHODS: This study was a self-controlled comparison study. Ten subjects diagnosed with superficial gastric neoplasia and scheduled to undergo endoscopic submucosal dissection (ESD) at a tertiary hospital were prospectively invited for an MCCG examination. The diagnostic agreement of MCCG, ESD and pathology were compared, including location, size and endoscopic appearance of the lesions. RESULTS: Of the 10 enrolled patients, 6 were confirmed as having early gastric cancer/high-grade intraepithelial neoplasia, 2 gastric low-grade intraepithelial neoplasia (LGIN), 1 tubular adenoma with LGIN and 1 neuroendocrine tumor. The per-patient and per-lesion sensitivities of MCCG for superficial gastric neoplasia detection were 100% and 91.7%. Location and size of the lesions were compared favorably to gastroscopy whilst one cardiac lesion was missed. Endoscopic appearances of these lesions observed on MCCG and EGD demonstrated good consistency. No adverse events were observed. CONCLUSION: With good gastric preparation and careful examination of stomach, MCCG is able to detect superficial gastric neoplasms.

Gastric preparation for magnetically controlled capsule endoscopy: A prospective, randomized single-blinded controlled trial.

BACKGROUND AND AIMS: Magnetically controlled capsule endoscopy (MCE) is a novel technique for which there is no agreed gastric preparation. We aimed to determine an optimal standardized gastric preparation regimen. METHODS: 120 patients referred for MCE were randomly assigned to gastric preparation with either water alone (A), water with simethicone (B) or water, simethicone and pronase (C). Image quality was assessed using cleanliness and visualization scores, higher scores equating to better image quality. RESULTS: The total cleanliness scores were (mean±SD) 15.83±2.41 (A), 21.35±1.23 (B), and 20.82±1.90 (C). The total visualization scores (mean±SD) were 10.75±2.02 (A), 15.20±1.32 (B), and 15.08±1.86 (C). While the image quality of the whole stomach in groups B and C were significantly better than group A (P<0.0001), there was no statistical difference between group B and C (P>0.05). MCE detected positive findings in 21 (52.5%), 27 (67.5%) and 21 (53.8%) patients in group A, B and C respectively, with no significant difference between groups (P>0.5). CONCLUSIONS: Simethicone swallowed with water prior to MCE produced the optimal gastric mucosal image quality. The addition of pronase had no demonstrable additional benefit.

Safety and Efficacy of a New Smartphone-controlled Vibrating Capsule on Defecation in Beagles.

Constipation, mainly manifesting as abdominal discomfort and painful defecation, is considered as a chronic disorder. Due to a lack of effective therapy, it imposes a significant economic burden and greatly impacts patients' quality of life which prompt searches for new, original approaches. Based on the research of vibrating capsule (VC) carried out by Ron et al., we investigated the safety and efficacy of an innovative, multi-mode VC in terms of its effect on defecation in animal studies. The parameters associated with different operation modes of VCs can be detected and adjusted by smartphone controlled external configuration device (ECD). The results of blood tests, physiological parameters, CT scan and pathological examination showed no significant abnormality, which undoubtedly confirmed the safety of VCs. For efficacy studies, defecation frequency of beagles increased after administration of these capsules without influence on stool characters. Meanwhile, the mean time of capsule evacuation tended to be reduced while showing no significant difference between different modes. In summary, this study elucidates the safety and effectiveness of VC in prompting the passage of gastrointestinal walls thus greatly increasing the defecation frequency. This study innovatively displays the promising application of VC in the treatment of constipation.

Incidence and risk factors for post-ERCP pancreatitis in chronic pancreatitis.

BACKGROUND AND AIMS: Almost all studies on post-ERCP pancreatitis (PEP) have mainly involved patients with biliary diseases rather than chronic pancreatitis (CP), and the concept that CP seems to be a protective factor associated with PEP has not been studied in detail. The aim of this study was to determine the incidence of PEP in patients with CP at different clinical stages and to identify the predictive and protective factors of PEP in a large cohort. METHODS: In this observational cohort study, medical records of patients with CP (CP group) and biliary diseases (BD group) in a tertiary hospital from January 2011 to May 2015 were examined. The difference in the incidence of PEP between CP group and BD group and the risk of PEP at different clinical stages of CP were calculated by the χ2 test or the Fisher exact test. The predictive and protective factors for PEP were investigated by univariate and multivariate analysis. RESULTS: In total, 2028 ERCP procedures were performed in 1301 patients with CP and 2000 procedures in 1655 patients with BD. The overall incidence of PEP in CP group (4.5%) was similar to that in the BD group (4.8%; P = .747). However, CP patients had significantly lower rates of moderate and severe attacks (0% vs 1.3%, P < .01). According to the M-ANNHEIM classification, the PEP incidences of CP at stages 0, I, II, III, and IV were 4.4%, 5.1%, 3.8%, 2.0%, and 2.0%, respectively. CP patients at stage Ia had the highest PEP incidence (8.0%) among all CP patients, significantly higher than that at stages Ib + Ic (3.9%) and II (3.8%). Female gender, history of acute pancreatitis, and prior PEP were independent risk factors of PEP, whereas extracorporeal shock wave lithotripsy was a protective factor. CONCLUSIONS: Compared with BD patients, CP patients had similar incidence of PEP overall but lower grades of severity. The incidence of PEP in CP patients decreased significantly with disease progression. (Clinical trial registration number: NCT02781987.).

Late-stage inhibition of autophagy enhances calreticulin surface exposure.

Calreticulin (CRT) exposure on the cell surface is essential for inducing immunogenic cell death by chemotherapy. Recent studies have shown conflicting effects of chemotherapy-induced autophagy on CRT exposure in cancer cells. Our data revealed that surface-exposed CRT (Ecto-CRT) emission was attenuated by inhibition of autophagy at early stages; however, inhibition of autophagy at late stages resulted in increased Ecto-CRT. Furthermore, neither autophagy activation nor endoplasmic reticulum (ER) stress induction alone was sufficient for CRT surface exposure. Moreover, chemotherapeutic agents that only activated autophagy without inducing ER stress could not increase Ecto-CRT; therefore, combined use of an autophagy activator and ER stress inducer could effectively promote CRT translocation to the plasma membrane. Together, our results highlight the potential of the combined use of ER stress inducers and autophagy late-stage inhibitors to reestablish and strengthen both the CRT exposure and immunogenicity of chemotherapeutic agents induced death cells.

Sirtuin 1 (SIRT1) activation mediates sildenafil induced delayed cardioprotection against ischemia-reperfusion injury in mice.

BACKGROUND: It has been well documented that phosphodiesterase-5 inhibitor, sildenafil (SIL) protects against myocardial ischemia/reperfusion (I-R) injury. SIRT1 is part of the class III Sirtuin family of histone deacetylases that deacetylates proteins involved in cellular stress response including those related to I-R injury. OBJECTIVE/HYPOTHESIS: We tested the hypothesis that SIL-induced cardioprotection may be mediated through activation of SIRT1. METHODS: Adult male ICR mice were treated with SIL (0.7 mg/kg, i.p.), Resveratrol (RSV, 5 mg/kg, a putative activator of SIRT1 used as the positive control), or saline (0.2 mL). The hearts were harvested 24 hours later and homogenized for SIRT1 activity analysis. RESULTS: Both SIL- and RSV-treated mice had increased cardiac SIRT1 activity (P<0.001) as compared to the saline-treated controls 24 hours after drug treatment. In isolated ventricular cardiomyocytes, pretreatment with SIL (1 µM) or RSV (1 µM) for one hour in vitro also upregulated SIRT1 activity (P<0.05). We further examined the causative relationship between SIRT1 activation and SIL-induced late cardioprotection. Pretreatment with SIL (or RSV) 24 hours prior to 30 min ischemia and 24 hours of reperfusion significantly reduced infarct size, which was associated with a significant increase in SIRT1 activity (P<0.05). Moreover, sirtinol (a SIRT1 inhibitor, 5 mg/kg, i.p.) given 30 min before I-R blunted the infarct-limiting effect of SIL and RSV (P<0.001). CONCLUSION: Our study shows that activation of SIRT1 following SIL treatment plays an essential role in mediating the SIL-induced cardioprotection against I-R injury. This newly identified SIRT1-activating property of SIL may have enormous therapeutic implications.

Dietary inorganic nitrate alleviates doxorubicin cardiotoxicity: mechanisms and implications.

Doxorubicin (DOX) is one of the most powerful and widely prescribed chemotherapeutic agents to treat divergent human cancers. However, the clinical use of DOX is restricted due to its severe cardiotoxic side-effects. There has been ongoing search for cardioprotectants against DOX toxicity. Inorganic nitrate has emerged as a bioactive compound that can be reduced into nitrite and nitric oxide in vivo and in turn plays a therapeutic role in diseases associated with nitric oxide insufficiency or dysregulation. In this review, we describe a novel concept of using dietary supplementation of inorganic nitrate to reduce DOX-induced cardiac cellular damage and dysfunction, based on our recent promising studies in a mouse model of DOX cardiotoxicity. Our data show that chronic oral ingestion of sodium nitrate, at a dose equivalent to ~400% of the Acceptable Daily Intake of the World Health Organization, alleviated DOX-induced left ventricular dysfunction and mitochondrial respiratory chain damage. Such cardioprotective effects were associated with reduction of cardiomyocyte necrosis/apoptosis, tissue lipid peroxidation, and mitochondrial H(2)O(2) generation following DOX treatment. Furthermore, proteomic studies revealed enhanced cardiac expression of mitochondrial antioxidant enzyme - peroxiredoxin 5 in the nitrate-treated animals. These studies suggest that inorganic nitrate could be an inexpensive therapeutic agent for long-term oral administration in preventing DOX-induced cardiac toxicity and myopathy during the prolonged pathological process. Future clinical trials in the cancer patients undergoing DOX chemotherapy are warranted to translate these experimental findings into an effective new therapy in preventing the DOX-induced cardiomyopathy.

Type 2 diabetic obese db/db mice are refractory to myocardial ischaemic post-conditioning in vivo: potential role for Hsp20, F1-ATPase δ and Echs1.

Ischaemic post-conditioning (PostC) is a clinically relevant cardioprotective modality that has been confirmed in many species including human. It remains unknown if PostC can still protect heart in Type 2 diabetes, a rapidly growing disease in the world. This study investigated the efficacy of PostC in the leptin receptor-deficient db/db mice, which possess Type 2 diabetic characteristics including obesity, hyperglycaemia and hyperleptinaemia. Adult male C57BL/6J wild-type (WT) and db/db mice were anaesthetized, mechanically ventilated and subjected to left coronary artery occlusion for 30 min. followed by 24 hrs of reperfusion. For the PostC groups, the hearts underwent six cycles of 10 sec. of reperfusion and 10 sec. of re-occlusion at the onset of reperfusion. The mice were sacrificed at the end of 24 hrs reperfusion for infarct size measurement. PostC significantly reduced infarct size in WT mice (n = 6/group; P < 0.05), but not in the db/db mice. To identify alterations in protein expression by PostC, proteomic analyses were performed in the heart samples using two-dimensional differential in-gel electrophoresis with three CyDye labelling, followed by mass spectrometry. The results show that mitochondrial proteins (F(1)-ATPase γ and Echs1) were down-regulated by PostC in WT heart. Such change was absent in the db/db heart. On the other hand, PostC reduced Hsp20 in the diabetic heart. In summary, PostC fails to protect Type 2 diabetic mice against ischaemia-reperfusion injury. The potential protein targets for the loss of PostC may include F(1)-ATPase γ, Echs1 and Hsp20 that could regulate cellular ATP consumption/production and defense response to ischaemic stress.

Dietary nitrate supplementation protects against Doxorubicin-induced cardiomyopathy by improving mitochondrial function.

OBJECTIVES: The aim of this study was to test the hypothesis that long-term dietary nitrate supplementation protects against doxorubicin-induced cardiomyopathy by improving ventricular function and reducing mitochondrial respiratory chain damage. BACKGROUND: Doxorubicin is a powerful anthracycline antibiotic used to treat divergent human neoplasms. Its clinical use is limited because of severe cardiotoxic side effects. Dietary nitrate and nitrite are essential nutrients for maintenance of steady-state tissue levels of nitric oxide and may play a therapeutic role in diseases associated with nitric oxide insufficiency or dysregulation. Dietary nitrate and nitrite supplementation alleviates myocardial injury caused by ischemia-reperfusion and cardiac arrest-resuscitation. METHODS: Adult male CF-1 mice were given a single dose of doxorubicin (15 mg/kg intraperitoneally), and left ventricular contractile function was assessed 5 days later using both echocardiography and pressure-volume Millar catheterization. A nitrate supplementation regimen (1 g/l sodium nitrate in drinking water) was started 7 days before doxorubicin injection and continued thereafter. Cardiomyocyte necrosis and apoptosis, tissue lipid peroxidation, and plasma nitrate and nitrite levels were assessed. In addition, mitochondrial complex I activity, oxidative phosphorylation capacity, and hydrogen peroxide generation were determined in parallel experiments. RESULTS: Doxorubicin caused impairment of ventricular contractility and cell death, which were significantly reduced by nitrate supplementation (p < 0.05). These cardioprotective effects were associated with a significant decrease in tissue lipid peroxidation. Nitrate supplementation significantly preserved mitochondrial complex I activity and oxidative phosphorylation and attenuated hydrogen peroxide generation after doxorubicin treatment. CONCLUSIONS: Long-term oral intake of inorganic nitrate attenuates doxorubicin-induced ventricular dysfunction, cell death, oxidative stress, and mitochondrial respiratory chain damage. Nitrate could be a promising therapeutic agent against doxorubicin-induced cardiotoxicity.

Identification of protein targets underlying dietary nitrate-induced protection against doxorubicin cardiotoxicity.

We recently demonstrated protective effect of chronic oral nitrate supplementation against cardiomyopathy caused by doxorubicin (DOX), a highly effective anticancer drug. The present study was designed to identify novel protein targets related to nitrate-induced cardioprotection. Adult male CF-1 mice received cardioprotective regimen of nitrate (1 g NaNO(3) per litre of drinking water) for 7 days before DOX injection (15 mg/kg, i.p.) and continued for 5 days after DOX treatment. Subsequently the heart samples were collected for proteomic analysis with two-dimensional differential in-gel electrophoresis with 3 CyDye labelling. Using 1.5 cut-off ratio, we identified 36 proteins that were up-regulated by DOX in which 32 were completely reversed by nitrate supplementation (89%). Among 19 proteins down-regulated by DOX, 9 were fully normalized by nitrate (47%). The protein spots were further identified with Matrix Assisted Laser Desorption/Ionization-Time-of-Flight (MALDI-TOF)/TOF tandem mass spectrometry. Three mitochondrial antioxidant enzymes were altered by DOX, i.e. up-regulation of manganese superoxide dismutase and peroxiredoxin 3 (Prx3), and down-regulation of Prx5, which were reversed by nitrate. These results were further confirmed by Western blots. Nitrate supplementation also significantly improved animal survival rate from 80% in DOX alone group to 93% in Nitrate + DOX group 5 days after the DOX treatment. In conclusion, the proteomic analysis has identified novel protein targets underlying nitrate-induced cardioprotection. Up-regulation of Prx5 by nitrate may explain the observed enhancement of cardiac antioxidant defence by nitrate supplementation.

Long-acting phosphodiesterase-5 inhibitor tadalafil attenuates doxorubicin-induced cardiomyopathy without interfering with chemotherapeutic effect.

Doxorubicin (DOX) is one of the most effective anticancer drugs. However, its cardiotoxicity remains a clinical concern that severely restricts its therapeutic usage. We designed this study to investigate whether tadalafil, a long-acting phosphodiesterase-5 (PDE-5) inhibitor, protects against DOX-induced cardiotoxicity. We also sought to delineate the cellular and molecular mechanisms underlying tadalafil-induced cardioprotection. Male CF-1 outbred mice were randomized into three groups (n = 15-24/group) to receive either saline (0.2 ml i.p.), DOX (15 mg/kg, given by a single intraperitoneal injection), or tadalafil (4 mg/kg p.o. daily for 9 days) plus DOX. Left ventricular function was subsequently assessed by transthoracic echocardiography and Millar conductance catheter. Cardiac contractile function was impaired by DOX, and it was significantly improved by cotreatment with tadalafil. Tadalafil attenuated DOX-induced apoptosis and depletion of prosurvival proteins, including Bcl-2 and GATA-4, in myocardium. Cardiac oxidative stress was attenuated and antioxidant capacity was enhanced by tadalafil possibly via up-regulation of mitochondrial superoxide dismutase (MnSOD). Moreover, the tadalafil-treated group demonstrated increased cardiac cGMP level and protein kinase G (PKG) activity. Tadalafil did not interfere with the efficacy of DOX in killing human osteosarcoma cells in vitro or its antitumor effect in vivo in tumor xenograft model. We conclude that tadalafil improved left ventricular function and prevented cardiomyocyte apoptosis in DOX-induced cardiomyopathy through mechanisms involving up-regulation of cGMP, PKG activity, and MnSOD level without interfering with the chemotherapeutic benefits of DOX.

Genomic changes in regenerated porcine coronary arterial endothelial cells.

OBJECTIVE: Genomic changes were defined in cultures of regenerated porcine coronary endothelial cells to explain the alterations that underlie their dysfunction. METHODS AND RESULTS: Regeneration of the endothelium was triggered in vivo by endothelial balloon denudation. After 28 days, both left circumflex (native cells) and left anterior descending (regenerated cells) coronary arteries were dissected, their endothelial cells harvested, and primary cultures established. The basal cyclic GMP production was reduced in regenerated cells without significant reduction in the response to bradykinin and A23187. The mRNA expression levels in both native and regenerated cells were measured by microarray and RT-PCR. The comparison revealed genomic changes related to vasomotor control (cyclooxygenase-1, angiotensin II receptor), coagulation (F2 and TFPI), oxidative stress (Mn SOD, GPX3, and GSR), lipid metabolism (PLA2 and HPGD), and extracellular matrix (MMPs). A-FABP and MMP7 were induced by regeneration. RT-PCR revealed upregulation of A-FABP and downregulation of eNOS and TR. The differential gene expression profiles were confirmed at the protein level by Western blotting for eNOS, F2, Mn SOD, MMP7, and TR. CONCLUSIONS: Cultures from regenerated coronary endothelial cells exhibit genomic changes explaining endothelial dysfunction and suggesting facilitation of coagulation, lipid peroxidation, and extracellular matrix remodeling.

Paracrine effects of direct intramyocardial implantation of bone marrow derived cells to enhance neovascularization in chronic ischaemic myocardium.

OBJECTIVE: To determine the optimal bone marrow (BM) cell types, and their potential mechanisms of action for neovascularization in chronic ischaemic myocardium. METHODS AND RESULTS: The functional effects, angiogenic potential and cytokine expression of direct intramyocardial implantation of autologous BM CD31-positive endothelial progenitor cells (EPC, n=9), BM mononuclear cells (MNCs, n=9), and saline (n=9) were compared in a swine model of chronic ischaemic myocardium. Autologous BM cells were harvested and catheter-based electromechanical mapping-guided direct intramyocardial injection was performed to target ischaemic myocardium. After 12 weeks, injection of BM-MNC resulted in significant improvements in left ventricular dP/dt (+21+/-8%, P=0.032), left ventricular pressure (+17+/-4%, P=0.048) and regional microsphere myocardial perfusion over ischaemic endocardium (+74+/-28%, P<0.05) and epicardium (+73+/-29%, P<0.05). No significant effects were observed following injection of BM-EPC or saline. Capillary density (1132+/-69 versus 903+/-44 per mm(2), P=0.047) and expression of mRNA of vascular endothelial growth factor (VEGF, 32.3+/-5.6 versus 13.1+/-3.7, P<0.05,) and angiopoietin-2 (23.9+/-3.6 versus 13.7+/-3.1, P<0.05) in ischaemic myocardium was significantly greater in the BM-MNC group than the saline group. The capillary density in ischaemic myocardium demonstrated a significant positive correlation with VEGF expression (r=0.61, P<0.001). CONCLUSION: Catheter-based direct intramyocardial injection of BM-MNC enhanced angiogenesis more effectively than BM-EPC or saline, possibly via a paracrine effect, with increased expression of VEGF that subsequently improved cardiac performance of ischaemic myocardium.