Polyethyleneterephthalate provides superior retention of endothelial cells during shear stress compared to polytetrafluoroethylene and pericardium

Background

Polyethyleneterephthalate (PET) and polytetrafluoroethylene (PTFE) are polymers successfully used as large diameter arterial grafts for peripheral vascular surgery. However, these prosthetic grafts are rarely used for coronary bypass surgery because of their low patency rates. Endothelialisation of the lumenal surface of these materials may improve their patency. This study aimed to compare the endothelialisation of PET, PTFE and pericardium by examining their seeding efficiency over time and the effect of various shear stresses on retention of endothelial cells.

Methods


Ovine endothelial cells at 4 × 105 cells/cm2 were seeded onto PET, PTFE and pericardium, and cultured for 1–168 hours. Cell coverage was determined via en face immunocytochemistry and cell retention was quantified after being subjected to shear stresses ranging from 0.018 to 0.037 N/m2 for 15, 30 and 60 minutes.

Results

Endothelial cells adhered to all of the materials one hour post-seeding. PET exhibited better cell retention rate, ranging from 66.9 ± 5.6% at 0.018 N/m2 for 15 min to 44.7 ± 1.9% at 0.037 N/m2 for 60 minutes, when compared to PTFE and pericardium (p < 0.0001, three-way ANOVA).

Conclusion

PET shows superior retention of endothelial cells during shear stress compare to PTFE and pericardium.

Expression, localisation and hormone regulation of the human copper transporter hCTR1 in placenta and choriocarcinoma Jeg-3 cells

Copper is an essential trace element necessary for normal growth and development. During pregnancy, copper is transported from the maternal circulation to the fetus by mechanisms which have not been clearly elucidated. The copper uptake protein, hCTR1 is predicted to play a role in copper transport in human placental cells. This study has examined the expression and localisation of hCTR1 in human placental tissue and Jeg-3 cells. In term placental tissue the hCTR1 protein was detected as a 105 kDa protein, consistent with the size of a trimer which may represent the functional protein. A 95 kDa band, possibly representing the glycosylated protein, was also detected. hCTR1 was localised within the syncytiotrophoblast layer and the fetal vascular endothelial cells in the placental villi and interestingly was found to be localised toward the basal plasma membrane. It did not co-localise with either the Menkes or the Wilson copper transporting ATPases. Using the placental cell line Jeg-3, it was shown that the 35 kDa monomer was absent in the extracts of cells exposed to insulin, estrogen or progesterone and in cells treated with estrogen an additional 65 kDa band was detected which may correspond to a dimeric form of the protein. The 95 kDa band was not detected in the cultured cells. These results provide novel insights indicating that hormones have a role in the formation of the active hCTR1 protein. Furthermore, insulin altered the intracellular localisation of hCTR1, suggesting a previously undescribed role of this hormone in regulating copper uptake through the endocytic pathway.

Identification of a novel polymorphism in the 3'UTR of the L-arginine transporter gene SLC7A1 : contribution to hypertension and endothelial dysfunction

Background— Endothelial dysfunction because of reduced nitric oxide bioavailability is a key feature of essential hypertension. We have found that normotensive siblings of subjects with essential hypertension have impaired endothelial function accompanied by altered arginine metabolism.

Methods and Results— We have identified a novel C/T polymorphism in the 3′UTR of the principal arginine transporter, solute carrier family 7 (cationic amino acid transporter, y+ system), member 1 gene (SLC7A1). The minor T allele significantly attenuates reporter gene expression (P<0.01) and is impaired in its capacity to form DNA-protein complexes (P<0.05). In 278 hypertensive subjects the frequency of the T allele was 13.3% compared with 7.6% in 498 normotensive subjects (P<0.001). Moreover, the overall genotype distribution observed in hypertensives differed significantly from that in normotensives (P<0.001). To complement these studies, we generated an endothelial-specific transgenic mouse overexpressing l-arginine transporter SLC7A1. The Slc7A1 transgenic mice exhibited significantly enhanced responses to the endothelium-dependent vasodilator acetylcholine (−log EC50 for wild-type versus Slc7A1 transgenic: 6.87±0.10 versus 7.56±0.13; P<0.001). This was accompanied by elevated production of nitric oxide by isolated aortic endothelial cells.

Conclusions— The present study identifies a key, functionally active polymorphism in the 3′UTR of SLC7A1. As such, this polymorphism may account for the apparent link between altered endothelial function, l-arginine, and nitric oxide metabolism and predisposition to essential hypertension.

Coassembled nanostructured bioscaffold reduces the expression of proinflammatory cytokines to induce apoptosis in epithelial cancer cells

The local inflammatory environment of the cell promotes the growth of epithelial cancers. Therefore, controlling inflammation locally using a material in a sustained, non-steroidal fashion can effectively kill malignant cells without significant damage to surrounding healthy cells. A promising class of materials for such applications are the nanostructured scaffolds formed by epitope containing minimalist self-assembled peptides (SAPs), as they are bioactive on a cellular length scale, whilst presenting as an easily handled hydrogel. Here, we show that the assembly process distributes an anti-inflammatory polysaccharide, fuccoidan, localised to the nanofibers to function as an anti-inflammatory biomaterial for cancer therapy. We show that it supports healthy cells, whilst inducing apoptosis in cancerous endothelial cells, as demonstrated by the downregulation of the proinflammatory gene and protein expression pathways associated with epithelial cancer progression. Our findings highlight an innovative material approach with potential applications as local epithelial cancer immunotherapy and drug delivery vehicles.

Role of P-glycoprotein in limiting the brain penetration of glabridin, an active isoflavan from the root of Glycyrrhiza glabra.

Purpose. Glabridin is a major active constituent of Glycyrrhiza glabra which is commonly used in the treatment of cardiovascular and central nervous system (CNS) diseases. Recently, we have found that glabridin is a substrate of P-glycoprotein (PgP/MDR1). This study aimed to investigate the role of PgP in glabridin penetration across the blood–brain barrier (BBB) using several in vitro and in vivo models.
Materials and Methods. Cultured primary rat brain microvascular endothelial cells (RBMVECs) were used in the uptake, efflux and transcellular transport studies. A rat bilateral in situ brain perfusion model was used to investigate the brain distribution of glabridin. The brain and tissue distribution of glabridin in rats with or without coadministered verapamil or quinidine were examined with correction for the tissue residual blood. In addition, the brain distribution of glabridin in mdr1a(-/-) mice was compared with the wild-type mice. Glabridin in various biological matrices was determined by a validated liquid chromatography mass spectrometric method.
Results. The uptake and efflux of glabridin in cultured RBMVECs were ATP-dependent and significantly altered in the presence of a PgP or multi-drug resistance protein (Mrp1/2) inhibitor (e.g. verapamil or MK-571). A polarized transport of glabridin was found in RBMVEC monolayers with
facilitated efflux from the abluminal (BL) to luminal (AP) side. Addition of a PgP or Mrp1/2 inhibitor in both luminal and abluminal sides attenuated the polarized transport across RBMVECs. In a bilateral in situ brain perfusion model, the uptake of glabridin into the cerebrum increased from 0.42 T 0.09% at 1 min to 9.27 T 1.69% (ml/100 g tissue) at 30 min and was significantly greater than that for sucrose. Coperfusion of a PgP or Mrp1/2 inhibitor significantly increased the brain distribution of glabridin by 33.6j142.9%. The rat brain levels of glabridin were only about 27% of plasma levels when corrected by tissue residual blood and it was increased to up to 44% when verapamil or quinidine was coadministered. The area under the brain concentration-time curve (AUC) of glabridin in mdr1a(-/-) mice was 6.0-fold higher than the wild-type mice.
Conclusions. These findings indicate that PgP limits the brain penetration of glabridin through the BBB and PgP may cause drug resistance to glabridin (licorice) therapy for CNS diseases and potential drugglabridin interactions. However, further studies are needed to explore the role of other drug transporters (e.g. Mrp1-4) in restricting the brain penetration of glabridin.

Increased versican content is associated with tendinosis pathology in the patellar tendon of athletes with jumper

Expansion of the extracellular matrix is a prominent but poorly characterized feature of tendinosis. The present study aimed to characterize the extent and distribution of the large aggregating proteoglycan versican in patients with patellar tendinosis. We obtained tendon from tendinopathy patients undergoing debridement of the patellar tendon and from controls undergoing intramedullary tibial nailing. Versican content was investigated by Western blotting and immunohistochemistry. Microvessel thickness and density were determined using computer-assisted image analysis. Markers for smooth muscle actin, endothelial cells (CD31) and proliferating cells (Ki67) were examined immunohistochemically. Western blot analysis and immunohistochemical staining revealed elevated versican content in the proximal patellar tendon of tendinosis patients (P=0.042). Versican content was enriched in regions of fibrocartilage metaplasia and fibroblast proliferation, as well as in the perivascular matrix of proliferating microvessels and within the media and intima of arterioles. Microvessel density was higher in tendinosis tissue compared with control tissue. Versican deposition is a prominent feature of patellar tendinosis. Because this molecule is not only a component of normal fibrocartilagenous matrices but also implicated in a variety of soft tissue pathologies, future studies should further detail both pathological and adaptive roles of versican in tendons.

Dual mechanisms for nitric oxide control of large arteries in the estuarine crocodile crocodylus porosus

n reptiles, accumulating evidence suggests that nitric oxide (NO) induces a potent relaxation in the systemic vasculature. However, very few studies have examined the source from which NO is derived. Therefore, the present study used both anatomical and physiological approaches to establish whether NO-mediated vasodilation is via an endothelial or neural NO pathway in the large arteries of the estuarine crocodile Crocodylus porosus. Specific endothelial nitric oxide synthase (NOS) staining was observed in aortic endothelial cells following nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and endothelial NOS immunohistochemistry (IHC), suggesting that an endothelial NO pathway is involved in vascular control. This finding was supported by in vitro organ bath physiology, which demonstrated that the relaxation induced by acetylcholine (10-5 mol l-1) was abolished in the presence of the NOS inhibitor, N-omega-nitro-L-arginine (L-NNA; 10-4 mol l-1), the soluble guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10-5 mol l-1), or when the endothelium was removed. Interestingly, evidence for a neural NO pathway was also identified in large arteries of the crocodile. Neural NOS was located in perivascular nerves of the major blood vessels following NADPH-d histochemistry and neural NOS IHC and in isolated aortic rings, L-NNA and ODQ, but not the removal of the endothelium, abolished the relaxation effect of the neural NOS agonist, nicotine (3x10-4 mol l-1). Thus, we conclude that the large arteries of C. porosus are potentially regulated by NO-derived from both endothelial and neural NOS.

Substrates and inhibitors of human multidrug resistance associated proteins and the implications in drug development

Human contains 49 ATP-binding cassette (ABC) transporter genes and the multidrug resistance associated proteins (MRP1/ABCC1, MRP2/ABCC2, MRP3/ABCC3, MRP4/ABCC4, MRP5/ABCC5, MRP6/ABCC6, MRP7/ABCC10, MRP8/ABCC11 and MRP9/ABCC12) belong to the ABCC family which contains 13 members. ABCC7 is cystic fibrosis transmembrane conductance regulator; ABCC8 and ABCC9 are the sulfonylurea receptors which constitute the ATP-sensing subunits of a complex potassium channel. MRP10/ABCC13 is clearly a pseudo-gene which encodes a truncated protein that is highly expressed in fetal human liver with the highest similarity to MRP2/ABCC2 but without transporting activity. These transporters are localized to the apical and/or basolateral membrane of the hepatocytes, enterocytes, renal proximal tubule cells and endothelial cells of the blood-brain barrier. MRP/ABCC members transport a structurally diverse array of important endogenous substances and xenobiotics and their metabolites (in particular conjugates) with different substrate specificity and transport kinetics. The human MRP/ABCC transporters except MRP9/ABCC12 are all able to transport organic anions, such as drugs conjugated to glutathione, sulphate or glucuronate. In addition, selected MRP/ABCC members may transport a variety of endogenous compounds, such as leukotriene C(4) (LTC(4) by MRP1/ABCC1), bilirubin glucuronides (MRP2/ABCC2, and MRP3/ABCC3), prostaglandins E1 and E2 (MRP4/ABCC4), cGMP (MRP4/ABCC4, MRP5/ABCC5, and MRP8/ABCC11), and several glucuronosyl-, or sulfatidyl steroids. In vitro, the MRP/ABCC transporters can collectively confer resistance to natural product anticancer drugs and their conjugated metabolites, platinum compounds, folate antimetabolites, nucleoside and nucleotide analogs, arsenical and antimonial oxyanions, peptide-based agents, and in concert with alterations in phase II conjugating or biosynthetic enzymes, classical alkylating agents, alkylating agents. Several MRP/ABCC members (MRPs 1-3) are associated with tumor resistance which is often caused by an increased efflux and decreased intracellular accumulation of natural product anticancer drugs and other anticancer agents. Drug targeting of these transporters to overcome MRP/ABCC-mediated multidrug resistance may play a role in cancer chemotherapy. Most MRP/ABCC transporters are subject to inhibition by a variety of compounds. Based on currently available preclinical and limited clinical data, it can be expected that modulation of MRP members may represent a useful approach in the management of anticancer and antimicrobial drug resistance and possibly of inflammatory diseases and other diseases. A better understanding of their substrates and inhibitors has important implications in development of drugs for treatment of cancer and inflammation.

Endothelialisation and cell retention on gelation-chitosan coated electrospun polyurethane, poly (lactide-co-glycolide) and collagen-coated pericardium

Arterial bypass and heart valve replacements are two of the most common surgical treatments in cardiovascular surgery today. Currently, artificial materials are used as substitute for these cardiac tissues. However, these foreign materials do not have the ability to grow, repair or remodel and are thrombogenic, leading to stenosis. With the aid of tissue engineering, it is possible to develop functional identical copies of healthy heart valves and arteries, which are biocompatible. Although much effort has been made into this area, there are still inconsistencies with respect to
endothelialisation and cell retention on synthetic biological grafts. These variations may be attributed to differences in factors such as cell seeding density, incubation periods and effects of shear stress. In this study, we have compared the endothelialisation and cell retention between gelain chitosan-coated electrospun polyurethane (PU), poly (lactide co-glycolide) (PGA/PLA) and collagen-coated pericardium. Endothelial cells adhered to all of the materials as early as 1–day post seeding. After 7-day of seeding, the coverage on PU was almost 45% and that on PGA/PLA was about 25% and the least was on collagen-coated pericardium of approximately 15%. It was observed that the PU showed superior cell coverage and cell retention in comparison to the PGA/PLA and collagen-coated pericardium.

Temporal expression of heat shock proteins 60 and 70 at lesion-prone sites during atherogenesis in ApoE-deficient mice

In the study, we investigate whether the expressions of heat shock protein (hsp)60 (a potential autoantigen) and the stress-inducible form of cytoprotector hsp70 are correlated with the development of atherosclerotic lesions in the aortic tree of apolipoprotein E–deficient (apoE^-/-) mice. The apoE^-/- mouse model is advantageous because the stress-inducible form of hsp70 is not constitutively expressed in mice, unlike primates; hence, tissues under stress can be clearly defined. Both mammalian hsps were detected newly expressed (before mononuclear cell infiltration) on aortic valves and endothelia at lesion-prone sites of 3-week-old apoE^-/- mice. In 8- and 20-week-old mice, they were strongly and heterogeneously expressed in early to advanced fibrofatty plaques, with levels correlating with lesion severity. Expression was markedly downregulated in advanced collagenous, acellular, calcified plaques of 40- and 69-week-old mice and was absent in control aortas of normocholesterolemic wild-type (apoE^+/+) mice. Western blot analysis of tissue homogenates confirmed the temporal expression of the hsps. Double immunostaining revealed that both hsps were expressed by lesional endothelial cells, macrophages, smooth muscle cells, and CD3⁺ T lymphocytes. This study provides evidence that hsp60 and hsp70 are temporally expressed on all major cell types in lesion-prone sites during atherogenesis, suggesting that few cells escape the toxic environment of the atherosclerotic plaque.

Promises of nanotechnology for drug delivery to brain in neurodegenerative diseases

Brain is a delicate organ, isolated from general circulation and characterized by the presence of relatively impermeable endothelial cells with tight junctions, enzymatic activity and the presence of active efflux transporter mechanisms. These formidable obstacles often block drug delivery to the brain across the blood-brain barrier (BBB). Although several promising molecules have the potential in the in vitro settings but lack of in vivo response is probably because the molecule cannot reach the brain in a sufficient concentration. Drug delivery across the BBB is a major limitation in the treatment of central nervous system (CNS) disorders and CNS infections. This review deals with the role of nanobiotechnology in CNS drug delivery, in which three categories of carbon nanotubes, nanowires and nanoparticles (NPs) are explained. The small size of the NPs makes them an ideal choice to penetrate the BBB. Several mechanisms are involved in this process and various strategies are used. There are some concerns about the safety of NP entry in the brain that need to be resolved before human use. Although there is no approved nanotechnology-based CNS drug available the future for such neuro-nanobiotechnology based delivery system developments is promising.

Modulating the interaction of CXCR4 and CXCL12 by low-molecular-weight heparin inhibits hepatic metastasis of colon cancer

Liver metastasis is the major obstacle for prolonging the survival of colon cancer patients. Low-molecular-weight heparin (LMWH), a common drug for venous thromboembolism, has displayed beneficial effects in improving the survival of cancer patients, though the mechanism remains unclear. This study aimed to investigate the effects of LMWH on hepatic metastasis of colon cancer and its underlying molecular mechanism by targeting the interaction of the chemokine receptor CXCR4 and its ligand CXCL12 (formerly known as stromal cell-derived factor 1α, SDF-1α), as the CXCR4-CXCL12 axis has been shown to regulate the interaction of cancer cells and stroma. Experimental results revealed that LMWH (Enoxaparin, 3500-5500 Da) inhibited the CXCL12-stimulated proliferation, adhesion and colony formation of human colon cancer HCT-116 cells that highly expressed CXCR4. Interestingly, LMWH or an anti-CXCR4 blocking antibody diminished the migrating and invading abilities of HCT116 cells stimulated by the recombinant CXCL12 protein or liver homogenates which contained endogenous CXCL12 protein. Although LMWH did not significantly inhibit the growth of subcutaneous colon tumors, it significantly suppressed the formation of hepatic metastasis established by intrasplenic injection of colon cancer cells in nude Balb/c mice and also downregulated the expression of CXCL12 in hepatic sinusoidal endothelial cells. The results suggest that LMWH inhibits the formation of hepatic metastasis of colon cancer by disrupting the interaction of CXCR4 and CXCL12, supporting that perioperative administration of LMWH may help to prevent the seeding and subsequent growth of hepatic metastases of colon cancer cells.