Thrombospondin-1 Drives Cardiac Remodeling in Chronic Kidney Disease.

Patients with chronic kidney disease (CKD) face a high risk of cardiovascular disease. Previous studies reported that endogenous thrombospondin 1 (TSP1) involves right ventricular remodeling and dysfunction. Here we show that a murine model of CKD increased myocardial TSP1 expression and produced left ventricular hypertrophy, fibrosis, and dysfunction. TSP1 knockout mice were protected from these features. In vitro, indoxyl sulfate is driving deleterious changes in cardiomyocyte through the TSP1. In patients with CKD, TSP1 and aryl hydrocarbon receptor were both differentially expressed in the myocardium. Our findings summon large clinical studies to confirm the translational role of TSP1 in patients with CKD.

Novel Perspectives in Chronic Kidney Disease-Specific Cardiovascular Disease.

Chronic kidney disease (CKD) affects > 10% of the global adult population and significantly increases the risk of cardiovascular disease (CVD), which remains the leading cause of death in this population. The development and progression of CVD-compared to the general population-is premature and accelerated, manifesting as coronary artery disease, heart failure, arrhythmias, and sudden cardiac death. CKD and CV disease combine to cause multimorbid cardiorenal syndrome (CRS) due to contributions from shared risk factors, including systolic hypertension, diabetes mellitus, obesity, and dyslipidemia. Additional neurohormonal activation, innate immunity, and inflammation contribute to progressive cardiac and renal deterioration, reflecting the strong bidirectional interaction between these organ systems. A shared molecular pathophysiology-including inflammation, oxidative stress, senescence, and hemodynamic fluctuations characterise all types of CRS. This review highlights the evolving paradigm and recent advances in our understanding of the molecular biology of CRS, outlining the potential for disease-specific therapies and biomarker disease detection.

Management and outcomes of pregnant ICU patients with severe COVID-19 pneumonia in Qatar: A retrospective cohort study.

INTRODUCTION: Pregnant women are considered a high-risk group for COVID-19 due to their increased vulnerability to viral infections. The impact of COVID-19 on pregnant women is not well understood, and there is a need for data on managing severe COVID-19 in pregnant patients. This retrospective descriptive cohort study described the characteristics, hospital stay, interventions, and outcomes of pregnant patients admitted to the intensive care units (ICUs) with severe COVID-19 pneumonia in Qatar. METHODS: Data were collected from medical records and chart reviews of pregnant women admitted to Hamad Medical Corporation (HMC) with COVID-19 pneumonia from March 01, 2020, to July 31, 2021. The inclusion criteria encompassed pregnant women with a positive polymerase chain reaction (PCR) antigen test or radiological changes at admission, requiring respiratory support, and hospitalized for more than 24 hours. RESULTS: A total of 43 pregnant women were included in this study. Most patients were admitted during the first wave of the pandemic, with a median gestational age of 212 days [interquartile range 178-242 days] at presentation. The most common respiratory support methods were high-flow nasal cannula, non-invasive positive pressure ventilation, and invasive positive pressure ventilation. Convalescent plasma therapy was administered to 58% of patients, and tocilizumab was used in 28%. Renal replacement therapy was required by 4.6% of patients and 7% required extracorporeal membrane oxygenation. CONCLUSION: This study provides valuable insights into the impact of COVID-19 on pregnant patients admitted to the ICUs in Qatar. The results suggest that pregnant patients with COVID-19 pneumonia require close monitoring and appropriate interventions to minimize adverse outcomes for both mother and fetus. The data may contribute to future guidelines and management strategies for severe COVID-19 in pregnant patients.

A metabolic role for CD47 in pancreatic ß cell insulin secretion and islet transplant outcomes.

Diabetes is a global public health burden and is characterized clinically by relative or absolute insulin deficiency. Therapeutic agents that stimulate insulin secretion and improve insulin sensitivity are in high demand as treatment options. CD47 is a cell surface glycoprotein implicated in multiple cellular functions including recognition of self, angiogenesis, and nitric oxide signaling; however, its role in the regulation of insulin secretion remains unknown. Here, we demonstrate that CD47 receptor signaling inhibits insulin release from human as well as mouse pancreatic ß cells and that it can be pharmacologically exploited to boost insulin secretion in both models. CD47 depletion stimulated insulin granule exocytosis via activation of the Rho GTPase Cdc42 in ß cells and improved glucose clearance and insulin sensitivity in vivo. CD47 blockade enhanced syngeneic islet transplantation efficiency and expedited the return to euglycemia in streptozotocin-induced diabetic mice. Further, anti-CD47 antibody treatment delayed the onset of diabetes in nonobese diabetic (NOD) mice and protected them from overt diabetes. Our findings identify CD47 as a regulator of insulin secretion, and its manipulation in ß cells offers a therapeutic opportunity for diabetes and islet transplantation by correcting insulin deficiency.

Disease-modifying interactions between chronic kidney disease and osteoarthritis: a new comorbid mouse model.

OBJECTIVE: The prevalence of comorbid chronic kidney disease (CKD) and osteoarthritis (OA) is increasing globally. While sharing common risk factors, the mechanism and consequences of concurrent CKD-OA are unclear. The aims of the study were to develop a preclinical comorbid model, and to investigate the disease-modifying interactions. METHODS: Seventy (70) male 8-10 week-old C57BL/6 mice were subjected to 5/6 nephrectomy (5/6Nx)±destabilisation of medial meniscus (DMM) or sham surgery. OA pathology and CKD were assessed 12 weeks postinduction by blinded histology scoring, micro-CT, immunohistochemistry for osteoclast and matrix metalloproteinase (MMP)-13 activity, and serum analysis of bone metabolic markers. RESULTS: The 5/6Nx model recapitulated characteristic features of CKD, with renal fibrosis and deranged serum alkaline phosphatase, calcium and phosphate. There was no histological evidence of cartilage pathology induced by 5/6Nx alone, however, synovial MMP-13 expression and subchondral bone osteoclastic activity were increased (p<0.05), with accompanying reductions (p<0.05) in subchondral trabecular bone, bone volume and mineral density. DMM significantly (p<0.05) increased tibiofemoral cartilage damage, subchondral bone sclerosis, marginal osteophytes and synovitis, in association with increased cartilage and synovial MMP-13. DMM alone induced (p<0.05) renal fibrosis, proteinuria and increased (p<0.05) 5/6Nx-induced serum urea. However, DMM in 5/6Nx-mice resulted in significantly reduced (p<0.05) cartilage pathology and marginal osteophyte development, in association with reduced subchondral bone volume and density, and inhibition of 5/6Nx-induced subchondral bone osteoclast activation. CONCLUSION: This study assessed a world-first preclinical comorbid CKD-OA model. Our findings demonstrate significant bidirectional disease-modifying interaction between CKD and OA.

Involvement of PAR-2 in the Induction of Cell-Specific Matrix Metalloproteinase-2 by Activated Protein C in Cutaneous Wound Healing.

We previously reported that human keratinocytes express protease-activated receptor (PAR)-2 and play an important role in activated protein C (APC)-induced cutaneous wound healing. This study investigated the involvement of PAR-2 in the production of gelatinolytic matrix metalloproteinases (MMP)-2 and -9 by APC during cutaneous wound healing. Full-thickness excisional wounds were made on the dorsum of male C57BL/6 mice. Wounds were treated with APC on days 1, 2, and 3 post-wounding. Cultured neonatal foreskin keratinocytes were treated with APC with or without intact PAR-2 signalling to examine the effects on MMP-2 and MMP-9 production. Murine dermal fibroblasts from PAR-2 knock-out (KO) mice were also assessed. MMP-2 and -9 were measured via gelatin zymography, fluorometric assay, and immunohistochemistry. APC accelerated wound healing in WT mice, but had a negligible effect in PAR-2 KO mice. APC-stimulated murine cutaneous wound healing was associated with the differential and temporal production of MMP-2 and MMP-9, with the latter peaking on day 1 and the former on day 6. Inhibition of PAR-2 in human keratinocytes reduced APC-induced MMP-2 activity by 25~50%, but had little effect on MMP-9. Similarly, APC-induced MMP-2 activation was reduced by 40% in cultured dermal fibroblasts derived from PAR-2 KO mice. This study shows for the first time that PAR-2 is essential for APC-induced MMP-2 production. Considering the important role of MMP-2 in wound healing, this work helps explain the underlying mechanisms of action of APC to promote wound healing through PAR-2.

The Role of Matrix Proteins in Cardiac Pathology.

The extracellular matrix (ECM) and ECM-regulatory proteins mediate structural and cell-cell interactions that are crucial for embryonic cardiac development and postnatal homeostasis, as well as organ remodeling and repair in response to injury. These proteins possess a broad functionality that is regulated by multiple structural domains and dependent on their ability to interact with extracellular substrates and/or cell surface receptors. Several different cell types (cardiomyocytes, fibroblasts, endothelial and inflammatory cells) within the myocardium elaborate ECM proteins, and their role in cardiovascular (patho)physiology has been increasingly recognized. This has stimulated robust research dissecting the ECM protein function in human health and disease and replicating the genetic proof-of-principle. This review summarizes recent developments regarding the contribution of ECM to cardiovascular disease. The clear importance of this heterogeneous group of proteins in attenuating maladaptive repair responses provides an impetus for further investigation into these proteins as potential pharmacological targets in cardiac diseases and beyond.

Pancreatic adenocarcinoma preferentially takes up and is suppressed by synthetic nanoparticles carrying apolipoprotein A-II and a lipid gemcitabine prodrug in mice.

We hypothesised that synthetic HDL nanoparticles carrying a gemcitabine prodrug and apolipoprotein A-II (sHDLGemA2) would target scavenger receptor-B1 (SR-B1) to preferentially and safely deliver gemcitabine into pancreatic ductal adenocarcinoma (PDAC). We designed, manufactured and characterised sHDLGemA2 nanoparticles sized ~130 nm, incorporating 20 mol% of a gemcitabine prodrug within the lipid bilayer, which strengthens on adding ApoA-II. We measured their ability to inhibit growth in cell lines and cell-derived and patient-derived murine PDAC xenografts. Fluorescent-labelled sHDLGemA2 delivered gemcitabine inside xenografts. Xenograft levels of active gemcitabine after sHDLGemA2 were similar to levels after high-dose free gemcitabine. Growth inhibition in mice receiving 4.5 mg gemcitabine/kg/d, carried in sHDLGemA2, was equivalent to inhibition after high-dose (75 mg/kg/d) free gemcitabine, and greater than inhibition after low-dose (4.5 mg/kg/d) free gemcitabine. sHDLGemA2 slowed growth in semi-resistant cells and a resistant human xenograft. sHDLGemA2 targeted xenografts more effectively than sHDLGemA1. SR-B1 was over-expressed in PDAC cells and xenografts. Targeting by ApoA-II was suppressed by anti-SR-B1. Because sHDLGemA2 provided only ~6% of the free gemcitabine dose for an equivalent response, patient side effects can be greatly reduced, and the sHDLGemA2 concept should be developed through clinical trials.

CD47 Promotes Age-Associated Deterioration in Angiogenesis, Blood Flow and Glucose Homeostasis.

The aged population is currently at its highest level in human history and is expected to increase further in the coming years. In humans, aging is accompanied by impaired angiogenesis, diminished blood flow and altered metabolism, among others. A cellular mechanism that impinges upon these manifestations of aging can be a suitable target for therapeutic intervention. Here we identify cell surface receptor CD47 as a novel age-sensitive driver of vascular and metabolic dysfunction. With the natural aging process, CD47 and its ligand thrombospondin-1 were increased, concurrent with a reduction of self-renewal transcription factors OCT4, SOX2, KLF4 and cMYC (OSKM) in arteries from aged wild-type mice and older human subjects compared to younger controls. These perturbations were prevented in arteries from aged CD47-null mice. Arterial endothelial cells isolated from aged wild-type mice displayed cellular exhaustion with decreased proliferation, migration and tube formation compared to cells from aged CD47-null mice. CD47 suppressed ex vivo sprouting, in vivo angiogenesis and skeletal muscle blood flow in aged wild-type mice. Treatment of arteries from older humans with a CD47 blocking antibody mitigated the age-related deterioration in angiogenesis. Finally, aged CD47-null mice were resistant to age- and diet-associated weight gain, glucose intolerance and insulin desensitization. These results indicate that the CD47-mediated signaling maladapts during aging to broadly impair endothelial self-renewal, angiogenesis, perfusion and glucose homeostasis. Our findings provide a strong rationale for therapeutically targeting CD47 to minimize these dysfunctions during aging.

Biomimetic Gemcitabine-Lipid Prodrug Nanoparticles for Pancreatic Cancer.

Gemcitabine (Gem) is a key drug for pancreatic cancer, yet limited by high systemic toxicity, low bioavailability and poor pharmacokinetic profiles. To overcome these limitations, Gem prodrug amphiphiles were synthesised with oleyl, linoleyl and phytanyl chains. Self-assembly and lyotropic mesophase behaviour of these amphiphiles were examined using polarised optical microscopy and Synchrotron SAXS (SSAXS). Gem-phytanyl was found to form liquid crystalline inverse cubic mesophase. This prodrug was combined with phospholipids and cholesterol to create biomimetic Gem-lipid prodrug nanoparticles (Gem-LPNP), verified by SSAXS and cryo-TEM to form liposomes. In vitro testing of the Gem-LPNP in several pancreatic cancer cell lines showed lower toxicity than Gem. However, in a cell line-derived pancreatic cancer mouse model Gem-LPNP displayed greater tumour growth inhibition than Gem using a fraction (<6 %) of the clinical dose and without any systemic toxicity. The easy production, improved efficacy and low toxicity of Gem-LPNP represents a promising new nanomedicine for pancreatic cancer.

Blocking thrombospondin-1 signaling via CD47 mitigates renal interstitial fibrosis.

Acute kidney injury triggers a complex cascade of molecular responses that can culminate in maladaptive repair and fibrosis. We have previously reported that the matrix protein thrombospondin-1 (TSP1), binding its high affinity its receptor CD47, promotes acute kidney injury. However, the role of this pathway in promoting fibrosis is less clear. Hypothesizing that limiting TSP1-CD47 signaling is protective against fibrosis, we interrogated this pathway in a mouse model of chronic ischemic kidney injury. Plasma and renal parenchymal expression of TSP1 in patients with chronic kidney disease was also assessed. We found that CD47-/- mice or wild-type mice treated with a CD47 blocking antibody showed clear amelioration of fibrotic histological changes compared to control animals. Wild-type mice showed upregulated TSP1 and pro-fibrotic markers which were significantly abrogated in CD47-/- and antibody-treated cohorts. Renal tubular epithelial cells isolated from WT mice showed robust upregulation of pro-fibrotic markers following hypoxic stress or exogenous TSP1, which was mitigated in CD47-/- cells. Patient sera showed a proportionate correlation between TSP1 levels and worsening glomerular filtration rate. Immunohistochemistry of human kidney tissue demonstrated tubular and glomerular matrix localization of TSP1 expression in patients with CKD. These data suggest that renal tubular epithelial cells contribute to fibrosis by activating TSP1-CD47 signaling, and point to CD47 as a potential target to limit fibrosis following ischemic injury.

Using patient-derived xenograft models of colorectal liver metastases to predict chemosensitivity.

BACKGROUND: Few in vivo models for colorectal cancer have been demonstrated to show external validity by accurately predicting clinical patient outcomes. Patient-derived xenograft (PDX) models of cancer have characteristics that might provide a form of translational research leading to personalized cancer care. The aim of this pilot study was to assess the feasibility of using PDXs as a platform for predicting patient colorectal liver metastases responses, in this case by correlating PDX and patient tumor responses to either folinic acid, fluorouracil plus oxaliplatin or folinic acid, fluorouracil plus irinotecan-based regimens. METHODS: Sixteen patients underwent potentially curative resection of colorectal liver metastases, and tumors were grafted into NOD.CB17-Prkdcscid/Arc mice. Mice were divided into groups to determine relative tumor growth in response to treatment. Tumors were analyzed by immunohistochemistry for Ki67 and Excision repair cross-complementation group 1. RESULTS: An engraftment rate of 81% was achieved. Overall, there was a 67% positive match rate between eligible patient and PDX chemosensitivity profiles. There was a significant difference in relative decrease in Ki67 expression between sensitive/stable versus resistant PDXs for both treatment regimens. There was no statistically significant correlation between baseline ERCC1 expression and response to Oxaliplatin + 5-Fluorouracil in the PDXs. CONCLUSIONS: This pilot study supports the feasibility of using PDX models of advanced colorectal cancer in larger studies to potentially predict patient chemosensitivity profiles.

Nuclear Insulin-Like Growth Factor Binding Protein-3 As a Biomarker in Triple-Negative Breast Cancer Xenograft Tumors: Effect of Targeted Therapy and Comparison With Chemotherapy.

Triple-negative breast cancer (TNBC) typically has a worse outcome than other breast cancer subtypes, in part owing to a lack of approved therapeutic targets or prognostic markers. We have previously described an oncogenic pathway in basal-like TNBC cells, initiated by insulin-like growth factor binding protein-3 (IGFBP-3), in which the epidermal growth factor receptor (EGFR) is transactivated by sphingosine-1-phosphate (S1P) resulting from sphingosine kinase (SphK)-1 activation. Oncogenic IGFBP-3 signaling can be targeted by combination treatment with the S1P receptor modulator and SphK inhibitor, fingolimod, and the EGFR kinase inhibitor, gefitinib (F + G). However, the interaction of this treatment with chemotherapy has not been documented. Since we observed nuclear localization of IGFBP-3 in some TNBC tumors, this study aimed to evaluate the prognostic significance of nuclear IGFBP-3 in pre-clinical models of basal-like TNBC treated with F + G and doxorubicin. Orthotopic xenograft tumors were grown in nude mice from the human basal-like TNBC cell lines MDA-MB-468 and HCC1806, and were treated with gefitinib, 25 mg/Kg, plus fingolimod, 5 mg/Kg, 3-times weekly. In some studies, doxorubicin was also administered once weekly for 6 weeks. Tumor tissue proteins were quantitated by immunohistochemistry (IHC). Interaction between doxorubicin and F + G was also studied in proliferation assays in vitro. In both tumor models, tissue staining for IGFBP-3 was predominantly nuclear. Combination of F + G significantly enhanced mouse survival, decreased nuclear IGFBP-3 and Ki67 staining, and increased apoptosis (cleaved caspase-3) staining. Kaplan-Meier survival analysis showed that a high tumor IGFBP-3 IHC score (>median), like a high Ki67 score, was significantly associated with shorter survival time, whereas a high apoptosis score was associated with prolonged survival. Studied in vitro in both cell lines, low-dose doxorubicin that had little effect alone, strongly enhanced the cytostatic effect of low-dose F + G combination. However, in both in vivo models, doxorubicin at maximum-tolerated dose neither inhibited tumor growth when administered alone, nor enhanced the significant inhibitory effect of F + G. We conclude that doxorubicin may not add benefit to the inhibitory effect of F + G unless its dose-limiting toxicity can be overcome. Nuclear IGFBP-3 appears to have potential as a prognostic marker in TNBC and could be evaluated for clinical utility.

Inhibition of basal-like breast cancer growth by FTY720 in combination with epidermal growth factor receptor kinase blockade.

BACKGROUND: New molecular targets are needed for women with triple-negative breast cancer (TNBC). This pre-clinical study investigated the combination of the EGFR inhibitor gefitinib with the sphingosine kinase (SphK) inhibitor FTY720 (Fingolimod), aiming to block tumorigenic signaling downstream of IGFBP-3, which is abundantly expressed in basal-like TNBC. METHODS: In studies of breast cancer cell growth in culture, proliferation was monitored by IncuCyte live-cell imaging, and protein abundance was determined by western blotting. In vivo studies of mammary tumor growth used two models: orthotopic xenograft tumors derived from three basal-like TNBC cell lines, grown in immune-deficient mice, and syngeneic murine 4T1 tumors grown in immune-competent mice. Protein abundance in tumor tissue was assessed by immunohistochemistry. RESULTS: Quantitated by live-cell imaging, the inhibitor combination showed synergistic cytostatic activity in basal-like cell lines across several TNBC molecular subtypes, the synergy being decreased by IGFBP-3 downregulation. Suppression of the tumorigenic mediator CD44 by gefitinib was potentiated by FTY720, consistent with CD44 involvement in the targeted pathway. In MDA-MB-468 and HCC1806 orthotopic TNBC xenograft tumors in nude mice, the drug combination inhibited tumor growth and prolonged mouse survival, although this effect was not significant for the gefitinib-resistant cell line HCC70. Combination treatment of murine 4T1 TNBC tumors in syngeneic BALB/c mice was more effective in immune-competent than immune-deficient (nude) mice, and a relative loss of tumor CD3 (T-cell) immunoreactivity caused by FTY720 treatment alone was alleviated by the drug combination, suggesting that, even at an FTY720 dose causing relative lymphopenia, the combination is still effective in an immune-competent setting. Immunohistochemistry of xenograft tumors showed significant enhancement of caspase-3 cleavage and suppression of Ki67 and phospho-EGFR by the drug combination, but SphK1 downregulation occurred only in MDA-MB-468 tumors, so is unlikely to be integral to treatment efficacy. CONCLUSIONS: Our data indicate that targeting IGFBP-3-dependent signaling pathways through gefitinib-FTY720 co-therapy may be effective in many basal-like breast cancers, and suggest tissue IGFBP-3 and CD44 measurement as potential biomarkers of treatment efficacy.

Apolipoprotein A-II Plus Lipid Emulsion Enhance Cell Growth via SR-B1 and Target Pancreatic Cancer In Vitro and In Vivo.

BACKGROUND: Apolipoprotein A-II (ApoA-II) is down regulated in the sera of pancreatic ductal adenocarcinoma (PDAC) patients, which may be due to increase utilization of high density lipoprotein (HDL) lipid by pancreatic cancer tissue. This study examined the influence of exogenous ApoA-II on lipid uptake and cell growth in pancreatic cancer (PC) both in vitro and in vivo. METHODS: Cryo transmission electron microscopy (TEM) examined ApoA-II's influence on morphology of SMOFLipid emulsion. The influence of ApoA-II on proliferation of cancer cell lines was determined by incubating them with lipid+/-ApoA-II and anti-SR-B1 antibody. Lipid was labeled with the fluorophore, DiD, to trace lipid uptake by cancer cells in vitro by confocal microscopy and in vivo in PDAC patient derived xenograft tumours (PDXT) by fluorescence imaging. Scavenger receptor class B type-1(SR-B1) expression in PDAC cell lines and in PDAC PDXT was measured by western blotting and immunohistochemistry, respectively. RESULTS: ApoA-II spontaneously converted lipid emulsion into very small unilamellar rHDL like vesicles (rHDL/A-II) and enhanced lipid uptake in PANC-1, CFPAC-1 and primary tumour cells as shown by confocal microscopy. SR-B1 expression was 13.2, 10.6, 3.1 and 2.3 fold higher in PANC-1, MIAPaCa-2, CFPAC-1 and BxPC3 cell lines than the normal pancreatic cell line (HPDE6) and 3.7 fold greater in PDAC tissue than in normal pancreas. ApoA-II plus lipid significantly increased the uptake of labeled lipid and promoted cell growth in PANC-1, MIAPaCa-2, CFPAC-1 and BxPC3 cells which was inhibited by anti SR-B1 antibody. Further, ApoA-II increased the uptake of lipid in xenografts by 3.4 fold. CONCLUSION: Our data suggest that ApoA-II enhance targeting potential of lipid in pancreatic cancer which may have imaging and drug delivery potentialities.

A patient-derived subrenal capsule xenograft model can predict response to adjuvant therapy for cancers in the head of the pancreas.

BACKGROUND: Although gemcitabine is commonly used as adjuvant therapy for pancreatic adenocarcinoma and pancreaticobiliary-type periampullary cancers, not all patients appear to benefit. This translational study evaluates the potential of a patient-derived subrenal capsule pancreatic cancer xenograft (SRCPCX) model to identify within eight weeks after surgery those tumours which will respond to gemcitabine. METHODS: SRCPCXs from 32 pancreatectomy patients were established in six to ten NOD/SCID mice per patient. After four weeks the mice were randomly assigned to receive gemcitabine or saline for four more weeks. After eight weeks, gemcitabine response in the grafts was evaluated by the percentage of tumour growth inhibition (%TGI), histological morphology and immunohistochemical markers (Ki-67, CK7 and cleaved caspase-3). These were collated into an Overall Response. Survival was assessed by Kaplan-Meier and Cox multivariate analyses. RESULTS: 375 of 450 pieces of tissue from 27 of 31 patients were evaluable. In 90% of patients, histopathological and immunostaining features of saline-treated control grafts were concordant with their original tumours. At follow up, six of 15 patients whose tumours had an Overall Response to gemcitabine died, compared with ten of 12 whose tumours did not respond (P = 0.025, Fisher's exact test). This was associated with improved survival on Kaplan-Meier analysis (P = 0.013). Cox multivariate analysis indicated that Overall Response, stage and grade were independent predictors of survival. CONCLUSION: This SRCPCX model retains major histopathological and immunohistochemical characteristics of the original tumour and when a combination of measures is used, enables early assessment of tumour sensitivity to gemcitabine in pancreatic cancers.

Cotargeting of epidermal growth factor receptor and PI3K overcomes PI3K-Akt oncogenic dependence in pancreatic ductal adenocarcinoma.

PURPOSE: PI3K-Akt is overexpressed in 50% to 70% of pancreatic ductal adenocarcinoma (PDAC). The hypothesis of this study is that PI3K and EGFR coinhibition may be effective in PDAC with upregulated PI3K-Akt signaling. EXPERIMENTAL DESIGN: Multiple inhibitors were tested on five PDAC cell lines. EGFR inhibitor (EGFRi)-resistant cell lines were found to have significantly overexpressed AKT2 gene, total Akt, and pAkt. In vitro erlotinib-resistant (ER) cell models (BxPC-ER and PANC-ER) with highly constitutively active PI3K-Akt were developed. These and their respective parent cell lines were tested for sensitivity to erlotinib, IGFIR inhibitor NVP-AEW541 (AEW), and PI3K-alpha inhibitor NVP-BYL719 (BYL), alone or in combination, by RTK-phosphoarray, Western blotting, immunofluorescence, qRT-PCR, cell proliferation, cell cycle, clonogenic, apoptosis, and migration assays. Erlotinib plus BYL was tested in vivo. RESULTS: Erlotinib acted synergistically with BYL in BxPC-ER (synergy index, SI = 1.71) and PANC-ER (SI = 1.44). Treatment of ER cell lines showing upregulated PI3K-Akt with erlotinib plus BYL caused significant G1 cell-cycle arrest (71%, P < 0.001; 58%, P = 0.003), inhibition of colony formation (69% and 72%, both P < 0.001), and necrosis and apoptosis (75% and 53%, both P < 0.001), more so compared with parent cell lines. In primary patient-derived tumor subrenal capsule (n = 90) and subcutaneous (n = 22) xenografts, erlotinib plus BYL significantly reduced tumor volume (P = 0.005). Strong pEGFR and pAkt immunostaining (2+/3+) was correlated with high and low responses, respectively, to both erlotinib and erlotinib plus BYL. CONCLUSION: PDAC with increased expression of the PI3K-Akt pathway was susceptible to PI3K-EGFR coinhibition, suggesting oncogenic dependence. Erlotinib plus BYL should be considered for a clinical study in PDAC; further evaluation of pEGFR and pAkt expression as potential positive and negative predictive biomarkers is warranted.

Activated protein C inhibits proliferation and tumor necrosis factor α-stimulated activation of p38, c-Jun NH2-terminal kinase (JNK) and Akt in rheumatoid synovial fibroblasts.

Synovial fibroblast proliferation is a hallmark of the invasive pannus in the rheumatoid joint. Activated protein C (APC) is a natural anticoagulant that exerts antiinflammatory and cyto-protective effects in various diseases via endothelial protein C receptor (EPCR) and proteinase-activated receptor (PAR)-mediated pathways. In this study, we investigated the effect and the underlying cellular signaling mechanisms of APC on proliferation of human rheumatoid synovial fibroblasts (RSFs). We found that APC stimulated proliferation of mouse dermal fibroblasts (MDFs) and normal human dermal fibroblasts (HDFs) by up to 60%, but robustly downregulated proliferation of RSFs. APC induced the phosphorylation of extracellular signal-regulated protein kinase (ERK) and enhanced expression of p21 and p27 in a dose-dependent manner in RSFs. The latter effect was inhibited by pre-treatment with the ERK inhibitors PD98059 and U0126 but not by p38 inhibitor SB203580. In addition, APC significantly downregulated tumor necrosis factor (TNF)α-stimulated cell proliferation and activation of p38, c-Jun NH2-terminal kinase (JNK) and Akt in RSFs. These results provide the first evidence that APC selectively inhibits proliferation and the inflammatory signaling pathways of RSFs. Thus, APC may reduce synovial hyperplasia and pannus invasion in rheumatoid arthritis.

Optimizing the enzyme loading and incubation time in enzymatic hydrolysis of lignocellulosic substrates.

A mathematical model for costing enzymatic hydrolysis of lignocellulosics is presented. This model is based on three variable parameters describing substrate characteristics and three unit costs for substrate, enzymes and incubation. The model is used to minimize the cost of fermentable sugars, as intermediate products on the route to ethanol or other biorefinery products, by calculating optimized values of enzyme loading and incubation time. This approach allows comparisons between substrates, with processing conditions optimized independently for each substrate. Steam-exploded pine wood was hydrolyzed in order to test the theoretical relationship between sugar yield and processing conditions.

Thermodynamic analysis of a high-yield biochemical process for biofuel production.

This paper presents a thermodynamic analysis of a high-yield biochemical process for biofuel production from lignocelluosic biomass based on a previously proposed process. Unlike the standard biochemical process, which ferments sugar intermediates to ethanol, the process under consideration converts sugars to acetic acid which is esterified and hydrogenated to produce ethanol. This process has a significantly higher yield and produces no carbon dioxide. However, we find that the thermodynamic efficiency of the process is not increased in proportion to the yield gain. An additional survey of various biofuel production processes showed no direct correlation between yield and thermodynamic efficiency. This survey and the detailed thermodynamic analyses lead us to conclude that yield alone is an unreliable performance metric for biofuel technologies.