Research and clinical translation of trilayer stent-graft of expanded polytetrafluoroethylene for interventional treatment of aortic dissection.

The aortic dissection (AD) is a life-threatening disease. The transcatheter endovascular aortic repair (EVAR) affords a minimally invasive technique to save the lives of these critical patients, and an appropriate stent-graft gets to be the key medical device during an EVAR procedure. Herein, we report a trilayer stent-graft and corresponding delivery system used for the treatment of the AD disease. The stent-graft is made of nitinol stents with an asymmetric Z-wave design and two expanded polytetrafluoroethylene (ePTFE) membranes. Each of the inner and outer surfaces of the stent-graft was covered by an ePTFE membrane, and the two membranes were then sintered together. The biological studies of the sintered ePTFE membranes indicated that the stent-graft had excellent cytocompatibility and hemocompatibility in vitro. Both the stent-graft and the delivery system exhibited satisfactory mechanical properties and operability. The safety and efficacy of this stent-graft and the corresponding delivery system were demonstrated in vivo. In nine canine experiments, the blood vessels of the animals implanted with the stent-grafts were of good patency, and there were no thrombus and obvious stenosis by angiography after implantation for 6 months. Furthermore, all of the nine clinical cases experienced successful implantation using the stent-graft and its postrelease delivery system, and the 1-year follow-ups indicated the preliminary safety and efficacy of the trilayer stent-graft with an asymmetric Z-wave design for interventional treatment.

Association of matrix metalloprotease 1, 3, and 12 polymorphisms with rheumatic heart disease in a Chinese Han population.

BACKGROUND: Rheumatic heart disease (RHD) is an autoimmune disease triggered by acute rheumatic fever (ARF). Matrix metalloproteinases (MMPs) play an important role in the modulation of immune responses. The purpose of this study was to evaluate the association of MMP1, 3, and 12 promoter polymorphisms with RHD in a Han population in Southern China since the 3 genes are localized on the same chromosome and have a combined effect. METHODS: DNA samples were obtained from 90 adult patients with RHD and 90 control subjects. Polymorphisms in MMP1 (rs1799750), MMP3 (rs3025058), and MMP12 (rs2276109) were genotyped by direct sequencing. Differences in genotype and allele frequencies of these polymorphisms were compared between the cases and the controls using Unconditional logistic regression models and Chi-squared test. RESULTS: The 2G/2G genotype of rs1799750 in MMP1 was associated with a significantly higher risk of RHD when compared with the 1G/1G genotype (OR = 3.227; 95% CI:1.118-9.31; p = 0.03). The frequency of allele 2G was higher in patients with RHD compared to the controls (69.4% vs. 58.9%; p = 0.048) No significant differences in genotype and allele frequencies of rs3025058 in MMP3 and rs2276109 in MMP12 were found between the patients with RHD and the controls (p > 0.05). CONCLUSIONS: Our results suggest that rs1799750 in MMP1 might be a risk factor for RHD in a Han population in Southern China, and individuals carrying the 2G/2G genotype are likely more susceptible to RHD. In contrast, rs3025058 in MMP3 and rs2276109 in MMP12 might not contribute to the risk of developing RHD in this population. Further studies with larger samples and other ethnic populations are required to confirm these findings.

Effect of R119G Mutation on Human P5CR1 Dynamic Property and Enzymatic Activity.

Pyrroline-5-carboxylate reductase (P5CR1) is a universal housekeeping enzyme that catalyzes the reduction of Δ1-pyrroline-5-carboxylate (P5C) to proline with concomitant oxidation of NAD(P)H to NAD(P)+. The enzymatic cycle between P5C and proline is important for function in amino acid metabolism, apoptosis, and intracellular redox potential balance in mitochondria. Autosomal recessive cutis laxa (ARCL) results from a mutation in P5CR1 encoded by PYCR1. Specifically, the R119G mutation is reported to be linked to ARCL although it has not yet been characterized. We synthesized R119G P5CR1 and compared it to WT P5CR1. Foldx prediction of WT and R119G mutant P5CR1 protein stability suggests that the R119G mutation could significantly reduce protein stability. We also performed enzymatic activity assays to determine how the mutation impacts P5CR1 enzymatic function. The results of these experiments show that mutagenesis of R119 to G decreases P5CR1 catalytic efficiency for 3,4-dehydro-L-proline relative to WT. Mutagenesis and kinetic studies reveal that the activity of the mutant decreases as temperature increases from 5°C to 37°C, with almost no activity at 37°C, indicating that this mutation impairs P5CR1 function in vivo. Conversely, WT P5CR1 retains its activity after incubation at 37°C and has essentially no remaining activity at 75°C. Taken together, our experimental results indicate the R119G mutation could be an involving pathomechanism for ARCL.

Effect of the R119G mutation on human P5CR structure and its interactions with NAD: Insights derived from molecular dynamics simulation and free energy analysis.

Pyrroline-5-carboxylate reductase (P5CR), an enzyme with conserved housekeeping roles, is involved in the etiology of cutis laxa. While previous work has shown that the R119G point mutation in the P5CR protein is involved, the structural mechanism behind the pathology remains to be elucidated. In order to probe the role of the R119G mutation in cutis laxa, we performed molecular dynamics (MD) simulations, essential dynamics (ED) analysis, and Molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) binding free energy calculations on wild type (WT) and mutant P5CR-NAD complex. These MD simulations and ED analyses suggest that the R119G mutation decreases the flexibility of P5CR, specifically in the substrate binding pocket, which could decrease the kinetics of the cofactor entrance and egress. Furthermore, the MM-PBSA calculations suggest the R119G mutant has a lower cofactor binding affinity for NAD than WT. Our study provides insight into the possible role of the R119G mutation during interactions between P5CR and NAD, thus bettering our understanding of how the mutation promotes cutis laxa.

In silico screening, molecular docking, and molecular dynamics studies of SNP-derived human P5CR mutants.

Pyrroline-5-carboxylate reductase (P5CR) encoded by PYCR1 gene is a housekeeping enzyme that catalyzes the reduction of P5C to proline using NAD(P)H as the cofactor. In this study, we used in silico approaches to examine the role of nonsynonymous single-nucleotide polymorphisms in the PYCR1 gene and their putative functions in the pathogenesis of Cutis Laxa. Among the 348 identified SNPs, 15 were predicted to be potentially damaging by both SIFT and PolyPhen tools; of them two SNP-derived mutations, R119G and G206W, have been previously reported to correlate with Cutis Laxa. These two mutations were therefore selected to be mapped to the wild-type (WT) P5CR structure for further structural and functional analyses. The results of comparative computational analyses using I-Mutant and Autodock reveal reductions in both stability and cofactor binding affinity of these two mutants. Comparative molecular dynamics (MD) simulations were performed to evaluate the changes in dynamic properties of P5CR upon mutations. The results reveal that the two mutations enhance the rigidity of P5CR structure, especially that of cofactor binding site, which could result in decreased kinetics of cofactor entrance and egress. Comparison between the structural properties of the WT and mutants during MD simulations shows that the enhanced rigidity of mutants results most likely from the increased number of inter-atomic interactions and the decreased number of dynamic hydrogen bonds. Our study provides novel insight into the deleterious effects of the R119G and G206W mutations on P5CR, and sheds light on the mechanisms by which these mutations mediate Cutis Laxa.

Physicochemical bases for protein folding, dynamics, and protein-ligand binding.

Proteins are essential parts of living organisms and participate in virtually every process within cells. As the genomic sequences for increasing number of organisms are completed, research into how proteins can perform such a variety of functions has become much more intensive because the value of the genomic sequences relies on the accuracy of understanding the encoded gene products. Although the static three-dimensional structures of many proteins are known, the functions of proteins are ultimately governed by their dynamic characteristics, including the folding process, conformational fluctuations, molecular motions, and protein-ligand interactions. In this review, the physicochemical principles underlying these dynamic processes are discussed in depth based on the free energy landscape (FEL) theory. Questions of why and how proteins fold into their native conformational states, why proteins are inherently dynamic, and how their dynamic personalities govern protein functions are answered. This paper will contribute to the understanding of structure-function relationship of proteins in the post-genome era of life science research.

Protein dynamics and motions in relation to their functions: several case studies and the underlying mechanisms.

Proteins are dynamic entities in cellular solution with functions governed essentially by their dynamic personalities. We review several dynamics studies on serine protease proteinase K and HIV-1 gp120 envelope glycoprotein to demonstrate the importance of investigating the dynamic behaviors and molecular motions for a complete understanding of their structure-function relationships. Using computer simulations and essential dynamic (ED) analysis approaches, the dynamics data obtained revealed that: (i) proteinase K has highly flexible substrate-binding site, thus supporting the induced-fit or conformational selection mechanism of substrate binding; (ii) Ca(2+) removal from proteinase K increases the global conformational flexibility, decreases the local flexibility of substrate-binding region, and does not influence the thermal motion of catalytic triad, thus explaining the experimentally determined decreased thermal stability, reduced substrate affinity, and almost unchanged catalytic activity upon Ca(2+) removal; (iii) substrate binding affects the large concerted motions of proteinase K, and the resulting dynamic pocket can be connected to substrate binding, orientation, and product release; (iv) amino acid mutations 375 S/W and 423 I/P of HIV-1 gp120 have distinct effects on molecular motions of gp120, facilitating 375 S/W mutant to assume the CD4-bound conformation, while 423 I/P mutant to prefer for CD4-unliganded state. The mechanisms underlying protein dynamics and protein-ligand binding, including the concept of the free energy landscape (FEL) of the protein-solvent system, how the ruggedness and variability of FEL determine protein's dynamics, and how the three ligand-binding models, the lock-and-key, induced-fit, and conformational selection are rationalized based on the FEL theory are discussed in depth.

Expression of matrix metalloproteinase-12 in aortic dissection.

BACKGROUND: Aortic dissection(AD) is an acute process of large blood vessels characterized by dangerous pathogenic conditions and high disability and high mortality. The pathogenesis of AD remains debated. Matrix metalloproteinase-12 (MMP-12) participates in many pathological processes such as abdominal aortic aneurysm, atherosclerosis, emphysema and cancer. However, this elastase has rarely been assessed in the presence of AD. The aim of the present study was to investigate the expression of MMP-12 in aortic tissue so as to offer a better understanding of the possible mechanisms of AD. METHODS: The protein expression levels of MMP-12 were analyzed and compared in aorta tissue and the blood serum samples by reverse transcription polymerase chain reaction(RT-PCR), Western blotting, immuno-histochemistry, fluorescence resonance energy transfer ( FRET ) activity assay and enzyme-linked immuno sorbent assay ( ELISA ), respectively. Ascending aorta tissue specimens were obtained from 12 patients with an acute Stanford A-dissection at the time of aortic replacement, and from 4 patients with coronary artery disease (CAD) undergoing coronary artery bypass surgery. Meanwhile, serum samples were harvested from 15 patients with an acute Stanford A-dissection and 10 healthy individuals who served as the control group. RESULTS: MMP-12 activity could be detected in both AD and CAD groups, but the level in the AD group was higher than those in the CAD group (P < 0.05). MMP-12 proteolysis existed in both serum samples of the AD and healthy groups, and the activity level in the AD group was clearly higher than in the healthy group (P < 0.05). For AD patients, MMP-12 activity in serum was higher than in the aorta wall (P < 0.05). MMP-12 activity in the aortic wall tissue can be inhibited by MMP inhibitor v (P < 0.05). CONCLUSION: The present study directly demonstrates that MMP-12 proteolytic activity exists within the aorta specimens and blood samples from aortic dissection patients. MMP-12 might be of potential relevance as a clinically diagnostic tool and therapeutic target in vascular injury and repair.