Identification of congenital aortic valve malformations in juvenile natriuretic peptide receptor 2-deficient mice using high-frequency ultrasound.

Mouse models of congenital aortic valve malformations are useful for studying disease pathobiology, but most models have incomplete penetrance [e.g., ∼2 to 77% prevalence of bicuspid aortic valves (BAVs) across multiple models]. For longitudinal studies of pathologies associated with BAVs and other congenital valve malformations, which manifest over months in mice, it is operationally inefficient, economically burdensome, and ethically challenging to enroll large numbers of mice in studies without first identifying those with valvular abnormalities. To address this need, we established and validated a novel in vivo high-frequency (30 MHz) ultrasound imaging protocol capable of detecting aortic valvular malformations in juvenile mice. Fifty natriuretic peptide receptor 2 heterozygous mice on a low-density lipoprotein receptor-deficient background (Npr2+/-;Ldlr-/-; 32 males and 18 females) were imaged at 4 and 8 wk of age. Fourteen percent of the Npr2+/-;Ldlr-/- mice exhibited features associated with aortic valve malformations, including 1) abnormal transaortic flow patterns on color Doppler (recirculation and regurgitation), 2) peak systolic flow velocities distal to the aortic valves reaching or surpassing ∼1,250 mm/s by pulsed-wave Doppler, and 3) putative fusion of cusps along commissures and abnormal movement elucidated by two-dimensional (2-D) imaging with ultrahigh temporal resolution. Valves with these features were confirmed by ex vivo gross anatomy and histological visualization to have thickened cusps, partial fusions, or Sievers type-0 bicuspid valves. This ultrasound imaging protocol will enable efficient, cost effective, and humane implementation of studies of congenital aortic valvular abnormalities and associated pathologies in a wide range of mouse models.NEW & NOTEWORTHY We developed a high-frequency ultrasound imaging protocol for diagnosing congenital aortic valve structural abnormalities in 4-wk-old mice. Our protocol defines specific criteria to distinguish mice with abnormal aortic valves from those with normal tricuspid valves using color Doppler, pulsed-wave Doppler, and two-dimensional (2-D) imaging with ultrahigh temporal resolution. This approach enables early identification of valvular abnormalities for efficient and ethical experimental design of longitudinal studies of congenital valve diseases and associated pathologies in mice.

Bioinspired Oligo-Urethane Nanoparticles for Delivering Exogenous C-Type Natriuretic Peptide: Synthetic Biomaterial Nanocarrier Complexes and Their Interactions with Cardiac Myofibroblasts.

In a healthy heart, cells naturally secrete C-type natriuretic peptide (CNP), a cytokine that protects against myofibroblast differentiation of cardiac fibroblasts and extracellular matrix deposition leading to fibrosis. CNP availability during myocardial remodeling is important to prevent cardiac fibrosis, but CNP is limited after an injury because of the loss of cardiomyocytes and the activation of cardiac fibroblasts to myofibroblasts. We hypothesized that the sustained release of exogenous CNP from oligo-urethane nanoparticles (NPs) would reduce differentiation of human cardiac fibroblasts toward a myofibrogenic phenotype. Our work used a modified form of a degradable polar hydrophobic ionic (D-PHI) oligo-urethane, which has shown the ability to self-assemble into NPs for the delivery of peptide and oligonucleotide biomolecules. The CNP-loaded NPs (NPCNP) were characterized for a diameter of 129 ± 1.4 nm and a ζ potential of -46 ± 7.8 mV. Treatment of cardiac fibroblasts with NPCNP increased cyclic guanosine-monophosphate (cGMP) synthesis, confirming that exogenous CNP delivered via oligo-urethane NPs is bioactive and can induce downstream signaling that has been implicated in antagonizing transforming growth factor-ß1 (TGF-ß1)-induced myofibrogenic differentiation. It is also shown that treatment with NPCNP attenuated contraction of collagen gels by cardiac myofibroblasts stimulated with TGF-ß1. Coating with heparin on the NPCNP (HEP-NPCNP) exemplified an approach to extend the release of CNP from the NPs. Both HEP-NPCNP and NPCNP show minimal cell toxicity, studied up to 0.25 × 1010 NPs/mL in culture media. These findings support further investigation of CNP delivery via NPs as a future therapy for suppressing cardiac fibrosis.

Association between multiple sclerosis and dietary patterns based on the traditional concept of food nature: a case-control study in Iran.

INTRODUCTION: It remains a matter of debate whether traditional concepts regarding the nature of food affect the development and progression of multiple sclerosis (MS).To date, there are limited studies that have investigated the association between MS and dietary patterns based on the categories of food nature (hot, cold, or balanced) defined in traditional medicine. METHOD: This case-control study was conducted from October 2019 to February 2020. In total, 60 patients diagnosed with MS within the preceding 6 months and referred to our neurology outpatient clinic were included in our case group. The control group included 180 patients who were referred to the same center for general or orthopedic surgery. Dietary intake was assessed in both groups through a reliable and valid semi-quantitative food frequency questionnaire. Data were assessed using principal component analysis. RESULTS: The mean age of the participants was 44.9 ± 17.33 years. The analysis showed that four food patterns were distinguished (eigenvalue > 1), namely "additives and cold-natured foods", "hot and balanced foods and nuts", "dairy and legumes", and "hot and balanced starches". These food patterns explained 57.8% of the total variance. After adjusting all confounding factors, individuals in the highest quartile and medium quartile of "additives and cold-natured foods" had an elevated MS risk compared with the lowest quartile (OR = 7.21, 95%CI = 2.01-12.38 and OR = 3.37, 95%CI = 1.02-11.35, respectively). Furthermore, individuals in the highest quartile of the "hot and balanced foods and nuts" group were protected against MS compared with its lowest quartile (OR = 0.28, 95%CI = 0.08-0.90). Moreover, a protective effect against MS was seen in the highest quartile of the "hot and balanced starches" group relative to its lowest quartile (OR = 0.34, 95%CI = 0.12-0.98). No significant association was found between "dairy and legumes" and the risk of MS. CONCLUSION: This study revealed that dietary patterns based on the traditional concept of food nature might be associated with the risk of developing MS. This represents the first work in this area, so further research is recommended.

Deficiency of Natriuretic Peptide Receptor 2 Promotes Bicuspid Aortic Valves, Aortic Valve Disease, Left Ventricular Dysfunction, and Ascending Aortic Dilatations in Mice.

RATIONALE: Aortic valve disease is a cell-mediated process without effective pharmacotherapy. CNP (C-type natriuretic peptide) inhibits myofibrogenesis and osteogenesis of cultured valve interstitial cells and is downregulated in stenotic aortic valves. However, it is unknown whether CNP signaling regulates aortic valve health in vivo. OBJECTIVE: The aim of this study is to determine whether a deficient CNP signaling axis in mice causes accelerated progression of aortic valve disease. METHODS AND RESULTS: In cultured porcine valve interstitial cells, CNP inhibited pathological differentiation via the guanylate cyclase NPR2 (natriuretic peptide receptor 2) and not the G-protein-coupled clearance receptor NPR3 (natriuretic peptide receptor 3). We used Npr2+/- and Npr2+/-;Ldlr-/- mice and wild-type littermate controls to examine the valvular effects of deficient CNP/NPR2 signaling in vivo, in the context of both moderate and advanced aortic valve disease. Myofibrogenesis in cultured Npr2+/- fibroblasts was insensitive to CNP treatment, whereas aged Npr2+/- and Npr2+/-;Ldlr-/- mice developed cardiac dysfunction and ventricular fibrosis. Aortic valve function was significantly impaired in Npr2+/- and Npr2+/-;Ldlr-/- mice versus wild-type littermates, with increased valve thickening, myofibrogenesis, osteogenesis, proteoglycan synthesis, collagen accumulation, and calcification. 9.4% of mice heterozygous for Npr2 had congenital bicuspid aortic valves, with worse aortic valve function, fibrosis, and calcification than those Npr2+/- with typical tricuspid aortic valves or all wild-type littermate controls. Moreover, cGK (cGMP-dependent protein kinase) activity was downregulated in Npr2+/- valves, and CNP triggered synthesis of cGMP and activation of cGK1 (cGMP-dependent protein kinase 1) in cultured porcine valve interstitial cells. Finally, aged Npr2+/-;Ldlr-/- mice developed dilatation of the ascending aortic, with greater aneurysmal progression in Npr2+/- mice with bicuspid aortic valves than those with tricuspid valves. CONCLUSIONS: Our data establish CNP/NPR2 signaling as a novel regulator of aortic valve development and disease and elucidate the therapeutic potential of targeting this pathway to arrest disease progression.

Psychometric properties of the Persian version of the Anesthetic Trainee Theatre Educational Environment Measure (ATEEM).

Background: The quality improvement of medical education programs and the ongoing reform of the curriculum should be done in the light of clinical training fields and identifying the strengths and improving the weaknesses. Therefore, this study was conducted to evaluate the validity and reliability of ATEEM (Anesthetic Trainee Theatre Educational Environment Measure) questionnaire for assessing learning environment of anesthesiology residents in educational centers affiliated to 3 main medical schools in Tehran, Iran. Methods: This study was conducted on first to fourth year anesthesiology residents using a questionnaire. Validity (face, content, construct) and reliability of ATEEM questionnaire was investigated. Construct validity was measured by confirmatory factor analysis, stability of reliability by test-retest, and internal consistency by Cronbach's alpha. Results: A total of 156 questionnaires out of 190 were fully answered, returned by residents of anesthesiology, and analysis were performed (82% response rate; 44.5% male (n=69); 55.5% female (n=86)). The age range of respondents was 26 to 48 years. The mean total ATEEM score was 114.03 out of 160. Face and content validity of the questionnaire was approved. Content validity ratio (CVR) and content validity index (CVI) were 0.63 and 0.88, respectively. Fitness indices in confirmatory factor analysis were greater than 0.9, and RMSEA (root mean square error of approximation) index was less than 0.08 (0.07). This indicator measures the acceptability of fitness and it is an appropriate measurement model. The average reliability coefficient was 0.73 and the overall Cronbach's alpha coefficient was 0.959. Conclusion: The results of this study showed that the Persian version of the ATEEM questionnaire, with appropriate psychometric properties, can be used to evaluate the anesthetic trainee theatre learning environment used in hospitals.

Positive-contrast cellular MRI of embryonic stem cells for tissue regeneration using a highly efficient T1 MRI contrast agent.

PURPOSE: To investigate the feasibility of high-sensitivity cellular MRI of embryonic stem (ES) cells using a novel cell permeable and cell retentive T1 contrast agent. MATERIALS AND METHODS: Mouse ES cells were labeled with a novel manganese porphyrin contrast agent, MnAMP, at 0.1 mM over 2 to 24 h and retained in contrast-free medium for up to 24 h postlabeling. MRI was performed on a 3 Tesla clinical scanner; T1 and T2 relaxation times were measured. Quantification of manganese content was performed using atomic absorption spectroscopy. Viability and proliferation assays were done for the longest labeling interval. Differentiation capacity was assessed using the hanging drop method to direct differentiation toward cardiomyocytes. RESULTS: MnAMP-labeled ES cells exhibited over a fourfold decrease in T1 compared with unlabeled cells, and maintained up to a threefold decrease 24 h postlabeling. Viability and proliferation were not affected. Most importantly, labeled ES cells differentiated into functional cardiomyocytes that exhibited normal contractility patterns. CONCLUSION: MnAMP-based cellular MRI is a very high sensitivity T1 approach for cellular imaging. It has the potential for noninvasive in vivo monitoring of stem cell therapy in cardiac regeneration and other tissue engineering and regenerative medicine applications. J. Magn. Reson. Imaging 2016;44:1456-1463.

Study of the influence of actin-binding proteins using linear analyses of cell deformability.

The actin cytoskeleton plays a key role in the deformability of the cell and in mechanosensing. Here we analyze the contributions of three major actin cross-linking proteins, myosin II, α-actinin and filamin, to cell deformability, by using micropipette aspiration of Dictyostelium cells. We examine the applicability of three simple mechanical models: for small deformation, linear viscoelasticity and drop of liquid with a tense cortex; and for large deformation, a Newtonian viscous fluid. For these models, we have derived linearized equations and we provide a novel, straightforward methodology to analyze the experiments. This methodology allowed us to differentiate the effects of the cross-linking proteins in the different regimes of deformation. Our results confirm some previous observations and suggest important relations between the molecular characteristics of the actin-binding proteins and the cell behavior: the effect of myosin is explained in terms of the relation between the lifetime of the bond to actin and the resistive force; the presence of α-actinin obstructs the deformation of the cytoskeleton, presumably mainly due to the higher molecular stiffness and to the lower dissociation rate constants; and filamin contributes critically to the global connectivity of the network, possibly by rapidly turning over cross-links during the remodeling of the cytoskeletal network, thanks to the higher rate constants, flexibility and larger size. The results suggest a sophisticated relationship between the expression levels of actin-binding proteins, deformability and mechanosensing.

Clinical profile of patients with seronegative celiac disease.

Aim: This study aimed to determine the clinical profile of patients with seronegative celiac disease (SNCD). Background: Celiac disease (CD) is mainly diagnosed based on positive serology and duodenal mucosal atrophy, but some patients have negative serology. Their diagnosis has some limitations; delays in diagnosis are likely accompanied by a poor prognosis and a high risk of developing complications of CD. Methods: In this retrospective study, 1115 patients were evaluated for CD with mucosal atrophy between 2010 to 2020. SNCD diagnosis requires genetic CD predisposition and improvement of both clinical symptoms and regrowth of duodenal villi after 12 months of a gluten-free diet (GFD) for all patients with IgA deficiency, other IgG-based serology for diagnosis of celiac was done and if these antibodies were negative, consider them as possible SNCD. If they had positive DQ2-DQ8 and improvement of clinical symptoms and mucosal atrophy after 12 months of GFD were confirmed SNCD. Results: Of the 1115 study subjects, 27 had SNCD, 1088 had SPCD with a mean age of 29.7±15.7 years (1 to 76 years) in seropositive celiac disease (SPCD) subjects and 37.1±16.3 years (6 to 63 years) in SNCD participants and 19 female patients with SNCD were presented. The BMI of SNCD and SPCD patients were reported 23.9 and 21.4, respectively. In addition, SPCD subjects were more likely but not statistically significant to have a positive family history. Villous atrophy was shown in 100% SNCD and 95.6% SPCD cases. Scalloping and fissuring in duodenal biopsies were reported in 60% of SNCD and 84.5% of SPCD patients. There was some other cause of seronegative villous atrophy including 3 patients with Crohns disease, 2 with common variable immunodeficiency, 2 drug and one patient with peptic duodenitis. Anemia, neurological symptoms, and liver function tests (LFT) abnormality were common extra intestinal manifestations in SNCD individuals. Levels of Thyroid peroxidase (TPO), TSH were measured, it had been detected that SNCD cases had a higher rate of co-occurrence with thyroid diseases also SPCD cases showed a higher rate of co-occurrence with diabetes. Conclusion: Among patients with celiac disease 2.4% are SNCD. SNCD are older than SPCD at the time of diagnosis and have higher BMI. Most common of cause of seronegative enteropathy also is SNCD followed by inflammatory bowel disease (IBD) common variable immunodeficiency (CVID), medication use, and duodenitis, in this area.

Serum- and xeno-free culture of human umbilical cord perivascular cells for pediatric heart valve tissue engineering.

BACKGROUND: Constructs currently used to repair or replace congenitally diseased pediatric heart valves lack a viable cell population capable of functional adaptation in situ, necessitating repeated surgical intervention. Heart valve tissue engineering (HVTE) can address these limitations by producing functional living tissue in vitro that holds the potential for somatic growth and remodelling upon implantation. However, clinical translation of HVTE strategies requires an appropriate source of autologous cells that can be non-invasively harvested from mesenchymal stem cell (MSC)-rich tissues and cultured under serum- and xeno-free conditions. To this end, we evaluated human umbilical cord perivascular cells (hUCPVCs) as a promising cell source for in vitro production of engineered heart valve tissue. METHODS: The proliferative, clonogenic, multilineage differentiation, and extracellular matrix (ECM) synthesis capacities of hUCPVCs were evaluated in a commercial serum- and xeno-free culture medium (StemMACS™) on tissue culture polystyrene and benchmarked to adult bone marrow-derived MSCs (BMMSCs). Additionally, the ECM synthesis potential of hUCPVCs was evaluated when cultured on polycarbonate polyurethane anisotropic electrospun scaffolds, a representative biomaterial for in vitro HVTE. RESULTS: hUCPVCs had greater proliferative and clonogenic potential than BMMSCs in StemMACS™ (p < 0.05), without differentiation to osteogenic and adipogenic phenotypes associated with valve pathology. Furthermore, hUCPVCs cultured with StemMACS™ on tissue culture plastic for 14 days synthesized significantly more total collagen, elastin, and sulphated glycosaminoglycans (p < 0.05), the ECM constituents of the native valve, than BMMSCs. Finally, hUCPVCs retained their ECM synthesizing capacity after 14 and 21 days in culture on anisotropic electrospun scaffolds. CONCLUSION: Overall, our findings establish an in vitro culture platform that uses hUCPVCs as a readily-available and non-invasively sourced autologous cell population and a commercial serum- and xeno-free culture medium to increase the translational potential of future pediatric HVTE strategies. This study evaluated the proliferative, differentiation and extracellular matrix (ECM) synthesis capacities of human umbilical cord perivascular cells (hUCPVCs) when cultured in serum- and xeno-free media (SFM) against conventionally used bone marrow-derived MSCs (BMMSCs) and serum-containing media (SCM). Our findings support the use of hUCPVCs and SFM for in vitro heart valve tissue engineering (HVTE) of autologous pediatric valve tissue. Figure created with BioRender.com.

Inhibition of pathological differentiation of valvular interstitial cells by C-type natriuretic peptide.

OBJECTIVE: Calcific aortic valve disease is associated with the differentiation of valvular interstitial cells (VICs) to myofibroblast and osteoblast-like cells, particularly in the fibrosa layer of the valve. Previous studies suggested that C-type natriuretic peptide (CNP) protects against calcific aortic valve disease to maintain homeostasis. We aimed to determine whether CNP inhibits VIC pathological differentiation as a mechanism to explain its protective effects. METHODS AND RESULTS: CNP expression was prominent in normal porcine aortic valves, particularly on the ventricular side, but reduced in sclerotic valves concomitant with the appearance of pathological VIC phenotypes in the fibrosa. In vitro, CNP inhibited calcified aggregate formation and bone-related transcript and protein expression by VICs grown in osteogenic conditions. Under myofibrogenic culture conditions, CNP reduced α-smooth muscle actin expression and cell-mediated gel contraction, indicating inhibition of myofibroblast differentiation. Similar to CNP, simvastatin inhibited VIC osteoblast and myofibroblast differentiation in vitro. Strikingly, simvastatin upregulated CNP expression in VICs cultured under myofibrogenic conditions, and small interfering RNA knockdown of natriuretic peptide receptor-b (a CNP receptor) significantly reduced the antifibrotic effect of simvastatin, suggesting that it acts in part via CNP/NPR-B autocrine/paracrine signaling. CONCLUSIONS: CNP inhibits myofibroblast and osteoblast differentiation of VICs and is responsible in part for inhibition of VIC myofibroblast differentiation by statins, suggesting novel mechanisms to explain the protective effect of CNP and the pleiotropic effects of statins in the aortic valve.

Identification and characterization of a Drosophila proteasome regulatory network.

Maintaining adequate proteasomal proteolytic activity is essential for eukaryotic cells. For metazoan cells, little is known about the composition of genes that are regulated in the proteasome network or the mechanisms that modulate the levels of proteasome genes. Previously, two distinct treatments have been observed to induce 26S proteasome levels in Drosophila melanogaster cell lines, RNA interference (RNAi)-mediated inhibition of the 26S proteasome subunit Rpn10/S5a and suppression of proteasome activity through treatment with active-site inhibitors. We have carried out genome array profiles from cells with decreased Rpn10/S5a levels using RNAi or from cells treated with proteasome inhibitor MG132 and have thereby identified candidate genes that are regulated as part of a metazoan proteasome network. The profiles reveal that the majority of genes that were identified to be under the control of the regulatory network consisted of 26S proteasome subunits. The 26S proteasome genes, including three new subunits, Ubp6p, Uch-L3, and Sem1p, were found to be up-regulated. A number of genes known to have proteasome-related functions, including Rad23, isopeptidase T, sequestosome, and the genes for the segregase complex TER94/VCP-Ufd1-Npl4 were also found to be up-regulated. RNAi-mediated inhibition against the segregase complex genes demonstrated pronounced stabilization of proteasome substrates throughout the Drosophila cell. Finally, transcriptional reporter assays and deletion mapping studies in Drosophila demonstrate that proteasome mRNA induction is dependent upon the 5' untranslated regions (UTRs). Transfer of the 5' UTR from the proteasome subunit Rpn1/S2 to a noninducible promoter was sufficient to confer transcriptional upregulation of the reporter mRNA after proteasome inhibition.

The Roles of Matrix Stiffness and ß-Catenin Signaling in Endothelial-to-Mesenchymal Transition of Aortic Valve Endothelial Cells.

Valve stiffening is a hallmark of aortic valve stenosis caused by excess extracellular matrix accumulation by myofibroblasts. We aimed to elucidate whether matrix stiffness regulates endothelial-to-mesenchymal transition (EndMT) of adult valvular endothelial cells (VECs) to myofibroblasts as a mechanism to further promote valve fibrosis. In addition, we specifically examined the role of the Wnt/ß-catenin signaling pathway in the development of myofibroblasts during EndMT, as Wnt/ß-catenin signaling has been implicated in EndMT during heart development, is reactivated in valve disease, and is required for mechanically-regulated myofibrogenesis of valve interstitial cells. Clonally derived porcine VECs were cultured on soft (5 kPa) or stiff (50 kPa) silicone Sylgard 527 substrates and treated with transforming growth factor (TGF)-ß1 to induce EndMT. Immunofluorescent staining revealed that TGF-ß1 preferentially promoted EndMT in VECs on stiffer substrates, evidenced by a decrease in the endothelial marker VE-cadherin and an increase in the myofibroblast marker α-smooth muscle actin (α-SMA). These changes were accompanied by ß-catenin nuclear localization both in vitro and in vivo, assessed by immunostaining. Degradation of ß-catenin with endostatin reduced VEC myofibroblast transition, as indicated by decreased α-SMA fiber expression. We conclude that TGF-ß1-induced EndMT in aortic VECs is dependent on matrix stiffness and Wnt/ß-catenin signaling promotes myofibrogenesis during EndMT.

Blood transfusion versus hydroxyurea in beta-thalassemia in Iran: a cost-effectiveness study.

INTRODUCTION: Thalassemia intermedia is a type of anemia which has several treatments modalities. We aimed to study the cost effectiveness of two treatments, including blood transfusion and hydroxyurea, in patients with beta-thalassemia intermedia in south of Iran referred to a referral center affiliated to Iran, Shiraz University of Medical Sciences in 2015. MATERIALS AND METHODS: This was a cost-effectiveness study which was conducted on 122 patients with beta-thalassemia intermedia. The indicator of effectiveness in this study was the reduction of growth disorder (normal BMI). Data analysis was done using SPSS 21, Excel 2010 and Treeage 2011. Finally, the one-way sensitivity analysis was performed to determine the robustness of the results. RESULTS: The average annual costs of blood transfusion and the use of hydroxyurea in 2015 were 20733.27 purchasing power parity (PPP)$ and 7040.29 PPP$, respectively. The effectiveness of blood transfusion was57.4% while in hydroxyurea group was 60.7%. CONCLUSION: The results showed that the cost effectiveness of using hydroxyurea was more than that of blood transfusion. Therefore, it is recommended that the use of hydroxyurea in the treatment of patients with beta-thalassemia intermedia would become the first priority, and more basic and supplementary insurance coverage for treating such patients using hydroxyurea should be considered.

Engineered beta-cells secreting dipeptidyl peptidase IV-resistant glucagon-like peptide-1 show enhanced glucose-responsiveness.

Type 2 diabetes is a polygenic disorder characterized by increased insulin resistance, and impaired insulin secretion leading to abnormalities of glucose and lipid metabolism. Reduced responsiveness of the beta-cells to glucose is a critical feature of this syndrome. Glucagon-like peptide 1, a product of the pro-glucagon gene makes beta-cells competent and has many other anti-diabetic properties. We speculated whether GLP-1-based gene therapy could be an approach for treatment of type 2 diabetes. We started with a clone of rat insulinoma cells (S4 cells), which showed reduced responsiveness to glucose in terms of insulin secretion. We transfected these cells with a plasmid encoding a mutated form of GLP-1 (GLP-1-Gly8), which is resistant to the degrading enzyme dipeptidyl-peptidase IV. Activity of secreted GLP-1-Gly8 was assayed using Chinese hamster lung fibroblasts (CHL) cells that expressed cloned GLP-1 receptor and that were transfected with CRE-Luc. Stable cell lines (Glipsulin cells) obtained by this means produced and stored immunoreactive GLP-1-Gly8. In addition to insulin, the Glipsulin cells secreted the GLP-1-Gly8. The secreted GLP-1-Gly8 was active as evidenced by the ability of the conditioned media to elevate cAMP levels in CHL cells expressing GLP-1 receptors. Glipsulin cells responded to glucose with a 6.8 fold increase in insulin secretion compared to a 2.2 fold increase in the control cells. Our results demonstrate that prolonged exposure to GLP-1-Gly8 secreted by increases glucose-responsiveness of these cells. We speculate that engineering GLP-1-Gly8 secretion by beta-cells is a potential gene therapeutic strategy to treat diabetes.

Evaluation of a porcine model of early aortic valve sclerosis.

BACKGROUND: Calcific aortic valve disease (CAVD) is associated with significant cardiovascular morbidity. While late-stage CAVD is well-described, early pathobiological processes are poorly understood due to the lack of animal models that faithfully replicate early human disease. Here we evaluated a hypercholesterolemic porcine model of early diet-induced aortic valve sclerosis. METHODS: Yorkshire swine were fed either a standard or high-fat/high-cholesterol diet for 2 or 5 months. Right coronary aortic valve leaflets were excised and analyzed (immuno)histochemically. RESULTS: Early human-like proteoglycan-rich onlays formed between the endothelial layer and elastic lamina in the fibrosa layer of valve leaflets, with accelerated formation associated with hypercholesterolemia (P<.05). Lipid deposition was more abundant in hypercholesterolemic swine (P<.001), but was present in a minority (28%) of onlays. No myofibroblasts, MAC387-positive macrophages, or fascin-positive dendritic cells were detected in 2-month onlays, with only scarce myofibroblasts present at 5 months. Cells that expressed osteochondral markers Sox9 and Msx2 were preferentially found in dense proteoglycan-rich onlays (P<.05) and with hypercholesterolemia (P<.05). Features of more advanced human CAVD, including calcification, were not observed in this necessarily short study. CONCLUSIONS: Early aortic valve sclerosis in hypercholesterolemic swine is characterized by the formation of proteoglycan-rich onlays in the fibrosa, which can occur prior to significant lipid accumulation, inflammatory cell infiltration, or myofibroblast activation. These characteristics mimic those of early human aortic valve disease, and thus the porcine model has utility for the study of early valve sclerosis.

The elastic properties of valve interstitial cells undergoing pathological differentiation.

Increasing evidence indicates that the progression of calcific aortic valve disease (CAVD) is influenced by the mechanical forces experienced by valvular interstitial cells (VICs) embedded within the valve matrix. The ability of VICs to sense and respond to tissue-level mechanical stimuli depends in part on cellular-level biomechanical properties, which may change with disease. In this study, we used micropipette aspiration to measure the instantaneous elastic modulus of normal VICs and of VICs induced to undergo pathological differentiation in vitro to osteoblast or myofibroblast lineages on compliant and stiff collagen gels, respectively. We found that VIC elastic modulus increased after subculturing on stiff tissue culture-treated polystyrene and with pathological differentiation on the collagen gels. Fibroblast, osteoblast, and myofibroblast VICs had distinct cellular-level elastic properties that were not fully explained by substrate stiffness, but were correlated with α-smooth muscle actin expression levels. C-type natriuretic peptide, a peptide expressed in aortic valves in vivo, prevented VIC stiffening in vitro, consistent with its ability to inhibit α-smooth muscle actin expression and VIC pathological differentiation. These data demonstrate that VIC phenotypic plasticity and mechanical adaptability are linked and regulated both biomechanically and biochemically, with the potential to influence the progression of CAVD.

Transgenic mouse models support HCR as an effector gene in the PSORS1 locus.

Genetic susceptibility for psoriasis is regulated to the greatest extent by the PSORS1 locus. Three psoriasis-associated susceptibility alleles have been identified within it, namely, HLACw6, HCR*WWCC and CDSN*5, but strong linkage disequilibrium between them has made it difficult to distinguish their individual genetic effects, and animal models to study their effects are not known. To study the function of HCR, we engineered transgenic mice with either a non-risk allele of HCR or the HCR*WWCC risk allele under the control of the cytokeratin-14 promoter. These choices were motivated by the apparently dominant effect of PSORS1 on psoriasis susceptibility and the physiological expression of HCR in basal keratinocytes. Transgenic mice appeared phenotypically normal and histologically their skin was indistinguishable from wild-type mice. Expression studies using Affymetrix arrays suggested that the HCR risk allele has specific functional consequences relevant to the pathogenesis of psoriasis. Comparison of gene expression changes between non-risk and risk allele mice revealed similarities to previous observations in human psoriatic skin, including upregulation of cytokeratins 6, 16 and 17 in risk allele mice. We also observed changes in the expression of genes associated with terminal differentiation and formation of the cornified cell envelope. Our results support the concept that HCR may constitute an essential gene in the PSORS1 locus. These observations are also compatible with a model that a susceptibility gene for psoriasis induces changes that are contributory but not sufficient by itself to produce the clinical phenotype.