Cost-effectiveness of Vascular Access for Haemodialysis: Arteriovenous Fistulas Versus Arteriovenous Grafts

BackgroundThe use of an arteriovenous fistula (AVF) for haemodialysis treatment may be associated with a high early failure rate, but usually good long-term patency, while using an arteriovenous graft (AVG) yields a lower early failure rate with worse long-term patency. The aim of this study was to calculate and compare the costs and outcome of AVF and AVG surgery in terms of early and long-term patencies.MethodsA decision tree and a Markov model were constructed to calculate costs and performance of AVFs and AVGs. The model was populated with a retrospective cohort of HD patients receiving their first VA. The outcomes were determined probabilistically with a 5-year follow-up.ResultsAVFs were usable for a mean (95% CI) of 28.5 months (24.6–32.5 months), while AVGs showed a patency of 25.5 months (20.0–31.2 months). The use of AVFs was the dominant type of VA and €631 could be saved per patient/per month patency compared to AVG use. Regardless of the willingness to pay, the use of AVFs yielded a higher probability of being cost-effective compared to AVGs.ConclusionsAVFs are more cost-effective than AVGs. Nonetheless, early failure rates significantly influence AVF performance and initiatives to reduce early failure can improve its cost-effectiveness.     

Clinical Trial of New Polyurethane Vascular Grafts for Hemodialysis: Compared with Expanded Polytetrafluoroethylene Grafts

Abstract: We developed a new polyurethane vascular access graft coated with gelatin and reinforced with knitted polyester fibers (PE-PEUG). Advantages over expanded-polytetrafluoroethylene graft (E-PTFEG) were previously reported in experimental studies. Between May 1990 and August 1992, 39 PE-PEUGs including 34 loop and 5 straight and 18 E-PTFEGs including 18 loop were implanted to create arteriovenous (AV) fistulas in a total of 52 adult patients on maintenance hemodialysis (HD). They were followed up until October 1994. Hemostasis on the suture line was achieved within 3 min in all patients implanted with PE-PEUGs. Bleeding from the needle holes of PE-PEUG stopped within 10 min with gentle finger pressure. Minimal local edema developed in only a few patients implanted with PE-PEUG while most patients implanted with E-PTFEG developed moderate lo cal edema. One seroma formation was found in an E-PTFE case. Aneurysmal dilatations were observed twice in a PE-PEUG patient 9 and 17 months after the implantation and once in a E-PTFEG patient 2 years after the implantation. The cumulative patency rate at 1 year in the PE-PEUG and E-PTFEG groups were 53.2 and 70.8%, respectively. Our clinical study showed that the PE-PEUG had several advantages over E-PTFEG: prompt hemostasis, no persistent edema and no formation of seroma, no change in elasticity, and sufficient mechanical strength. However, the cumulative patency rate was inferior to that with E-PTFEG implanted in our series. Further modifications are therefore necessary to improve the patency rate.     

Improved Healing of Small-Caliber,Long-Fibril Expanded Polytetrafluoroethylene Vascular Grafts by Covalent Bonding of Fibronectin

Purpose To evaluate the intermediate performance of small-caliber, long-fibril expanded polytetrafluoroethylene (ePTFE) vascular grafts pretreated with covalent bonding of fibronectin in dogs.Methods Small-caliber (4mm), long-fibril (60µm), ePTFE vascular grafts, 10cm in length, were pretreated by covalent bonding of fibronectin. Bilateral iliac grafting was done in dogs using a fibronectin-bonded graft on one side and a nonbonded control graft on the other side. The grafts were retrieved 12 weeks after implantation, and subjected to histomorphometric analysis.Results Although the patency rates of the fibronectin-bonded and control grafts were the same (3/7, 43%), the fibronectin-bonded grafts showed almost complete neointimal healing, whereas the nonbonded control grafts showed only partial neointimal healing, proximally and distally.Conclusions Small-caliber, long-fibril ePTFE vascular grafts with covalent bonding of fibronectin achieved almost complete neointimal healing by the time of retrieval at 12 weeks. This indicates that, with further modifications, our new technique for covalent bonding of fibronectin has great potential in the development of small-caliber arterial prosthetic grafts.     

Evaluation of Neointimal Hyperplasia on Tranilast-Coated Synthetic Vascular Grafts: An Experimental Study

Tranilast is an antiallergic drug that interferes with proliferation and migration of vascular smooth muscle cell induced by platelet-derived growth factor (PDGF) and transforming growth factor-β1 (TGF-β1). We investigated the local effect of tranilast on neointimal hyperplasia using tranilast-coated prosthetic grafts. The inner sides of the thin-walled polytetrafluoroethylene (PTFE) grafts were coated with chitosan and tranilast containing chitosan solution. Wistar albino rats (32) were used in the study. Patches (1 × 2 mm) for vascular grafts were prepared. Three groups were tested: group 1 (n = 12; tranilast coated), group 2 (n = 10; adhesive-only film-layer–coated), and group 3 (n = 10; normal ePTFE patch grafts sutured to the carotid arteries of the rats). Recipient sites of the carotid arteries were excised 4 weeks after surgery. All sections were examined histologically for graft patency, thrombus formation, and neointimal thickness. Expression of PDGF, fibroblast growth factor, and TGF-β1 on cross-sections of the neointima were evaluated by immunohistochemistry. No significant differences were found regarding mean neointimal thicknesses. PDGF and TGF-β-1 expressions were significantly lower in group 1. Although a decrease in local effect of tranilast was observed for growth factor expressions at a drug concentration of 0.05 mg/cm², a significant reduction in neointimal hyperplasia was not achieved. The coating concentration of 0.05 mg/cm² may have been too low to produce an antiproliferative effect. Given our promising results, further studies are recommended and planned using different drug concentrations and time intervals.     

Expression of Vascular Adhesion Molecules in Saphenous Vein Coronary Bypass Grafts

Background. Adhesion of blood elements to the endothelium is an important step in the development of vein graft disease. This study examines the expression of vascular adhesion molecules on explanted saphenous vein bypass grafts.

Methods. Immunocytochemical staining was performed using explanted saphenous vein grafts from 28 patients. Antibodies against the endothelial markers CD31, von Willebrand factor, intercellular adhesion molecule-1, vascular adhesion molecule-1, and E-selectin were used.

Results. Staining for CD31 and von Willebrand factor demonstrated the presence of endothelial cells in the lumen and the vasa vasorum. Expression of intercellular adhesion molecule-1 was variable between grafts, whereas vascular adhesion molecule-1 and E-selectin were almost always absent on the luminal endothelium. In contrast, the endothelium of the vasa vasorum stained positively for intercellular adhesion molecule-1 and vascular adhesion molecule-1, and was also seen on nonendothelial cells within the vessel wall. Expression of these adhesion molecules did not vary with the severity of vein graft disease.

Conclusions. This study highlights the blood vessels in the adventitia as possible sites for the adhesion and migration of cells into the vessel wall.     

Nanoparticle-Mediated Expression of an Angiogenic Inhibitor Ameliorates Ischemia-Induced Retinal Neovascularization and Diabetes-Induced Retinal Vascular Leakage

OBJECTIVE

The aim of the study is to evaluate the effect of nanoparticle-mediated gene delivery of angiogenic inhibitors on retinal inflammation, vascular leakage, and neovascularization in diabetic retinopathy.

RESEARCH DESIGN AND METHODS

An expression plasmid of plasminogen kringle 5 (K5), a natural angiogenic inhibitor, was encapsulated with poly(lactide-coglycolide) to form K5 nanoparticles (K5-NP). Expression of K5 was determined by Western blot analysis and immunohistochemistry, and retinal vascular leakage was measured by permeability assay. Retinal neovascularization was evaluated using fluorescein-angiography and counting preretinal vascular cells in rats with oxygen-induced retinopathy. Effects of K5-NP on retinal inflammation were evaluated in streptozotocin-induced diabetic rats by leukostasis assay and Western blot analysis of intracellular adhesion molecule and vascular endothelial growth factor. Possible toxicities of K5-NP were evaluated using histology examination, retinal thickness measurement, and electroretinogram recording.

RESULTS

K5-NP mediated efficient expression of K5 and specifically inhibited growth of endothelial cells. An intravitreal injection of K5-NP resulted in high-level expression of K5 in the inner retina of rats during the 4 weeks they were analyzed. Injection of K5-NP significantly reduced retinal vascular leakage and attenuated retinal neovascularization, when compared with the contralateral eyes injected with Control-NP in oxygen-induced retinopathy rats. K5-NP attenuated vascular endothelial growth factor and intracellular adhesion molecule-1 overexpression and reduced leukostasis and vascular leakage for at least 4 weeks after a single injection in the retina of streptozotocin-induced diabetic rats. No toxicities of K5-NP were detected to retinal structure and function.

CONCLUSIONS

K5-NP mediates efficient and sustained K5 expression in the retina and has therapeutic potential for diabetic retinopathy.Retinal vascular leakage and neovascularization are the major features of diabetic retinopathy and the leading causes of vision loss (1–3). These retinal vascular abnormalities are also common in other ocular disorders such as sickle cell retinopathy, retinal vein occlusion, and retinopathy of prematurity (1,4,5). Vascular endothelial growth factor (VEGF) plays a key pathogenic role in the blood-retinal barrier breakdown or vascular leakage and retinal neovascularization (6–8).Angiogenesis is regulated by two counter-balancing systems between angiogenic stimulators, such as VEGFs and angiogenic inhibitors such as angiostatin (4,9,10). Angiostatin contains the first four triple disulfide bond-linked loops of plasminogen known as kringle domains and is a potent inhibitor of angiogenesis (11). Among proteolytic fragments of plasminogen, kringle 5 (K5), an 80–amino acid peptide from plasminogen, has the most potent inhibitory effect on endothelial cell growth (12). Previously, we have shown that K5 inhibits ischemia-induced retinal neovascularization in the oxygen-induced retinopathy (OIR) model (13). K5 also reduces retinal vascular leakage in the OIR model and in the streptozotocin (STZ)-induced diabetes model (14). The K5-induced reduction of vascular leakage is achieved through an intraocular, periocular, topical, or systemic administration of the K5 peptide (15). Similar to that of many other anti-angiogenic peptides, however, these K5 effects are transient after a single injection of the K5 peptide because of its short half-life in the retina (14,15). A sustained ocular delivery of K5, such as gene therapy, is desirable for the development of a long-term treatment of diabetic retinopathy.Traditionally, gene delivery systems can be classified into viral vector-mediated and nonviral deliveries. Currently, viral vectors are the most commonly used means for gene delivery because of their high efficiencies (16,17). The limitations of viral vector-mediated delivery, such as potential risks, restricted targeting of specific cell types, and immunogenecity of viral vectors hamper their clinical application (18,19). For these reasons, nonviral systems for gene delivery have become increasingly desirable in both basic research and clinical settings.One of the emerging nonviral vector-mediated gene delivery systems is condensation of plasmid DNA or oligonucleotides into nanoparticles (20). There are several biocompatible polymers to be used for DNA delivery, such as poly(d,l-lactide-coglycolide) (PLGA) and poly(ethylene-covinyl acetate) (EVAc). Several groups have successfully encapsulated naked DNA into biodegradable PLGA nanoparticles for long-term and controlled DNA release (21). Although matrix-type nanoparticles have been formulated using different polymers, the nanoparticles formulated from PLGA are especially of interest for gene delivery because of their safety, biocompatibility, biodegradability, and sustained release characteristics (22,23).We encapsulated an expression plasmid of K5 with PLGA polymer to form nanoparticles and evaluated the efficacy of these K5 nanoparticles (K5-NP) on ischemia-induced retinal vascular leakage and retinal neovascularization in the OIR rat model. We evaluated the effects of K5-NP on retinal inflammation in STZ-induced diabetic rats. In addition, we also tested the ocular toxicities of K5-NP.     

CKD Accelerates Development of Neointimal Hyperplasia in Arteriovenous Fistulas

Arteriovenous (AV) access failure resulting from venous neointimal hyperplasia is a major cause of morbidity in patients with ESRD. To understand the role of chronic kidney disease (CKD) in the development of neointimal hyperplasia, we created AV fistulae (common carotid artery to jugular vein in an end-to-side anastomosis) in mice with or without CKD (renal ablation or sham operation). At 2 and 3 wk after operation, neointimal hyperplasia at the site of the AV anastomosis increased 2-fold in animals with CKD compared with controls, but cellular proliferation in the neointimal hyperplastic lesions did not significantly differ between the groups, suggesting that the enhanced neointimal hyperplasia in the setting of CKD may be secondary to a migratory phenotype of vascular smooth muscle cells (VSMC). In ex vivo migration assays, aortic VSMC harvested from mice with CKD migrated significantly greater than VSMC harvested from control mice. Moreover, animals with CKD had higher serum levels of osteopontin, which stimulates VSMC migration. When we treated animals with bone morphogenic protein-7, which promotes VSMC differentiation, before creation of the AV anastomosis, the effect of CKD on the development of neointimal hyperplasia was eliminated. In summary, CKD accelerates development of neointimal hyperplasia at the anastomotic site of an AV fistula, and administration of bone morphogenic protein-7 neutralizes this effect.Arteriovenous (AV) access dysfunction such as stenosis and thrombosis constitute a major cause of morbidity for patients on chronic hemodialysis for end-stage kidney disease.¹ While AV fistulae constructed with native vessels are the best vascular access available owing to a lower incidence of stenosis, thrombosis, and infection compared with vascular grafts or central venous catheters, its failure rate up to 66% at 2 yr² remains unacceptably high as hemodialysis access related hospitalizations are on the rise and its cost are well over one billion dollars per annum in the United States alone.³The cause of failure is predominantly secondary to the occlusive neointimal hyperplastic (NH) lesion formation at the anastomosis and/or the outflow veins followed by in situ thrombosis.^4–7 Unlike restenosis seen with preocclusive atherosclerotic arteries after angioplasty and stenting, neointimal (new intimal) hyperplasia is seen at the anastomosis involving an artery or a synthetic graft (e.g., expanded polytetrafluoroethylene, or ePTFE, or Dacron) and a vein in the upper extremities. Although these blood vessels are predisposed to calcification, pre-existing NH, and needle stick injury, they are usually free of atherosclerotic plaque. Therefore, directional migration of vascular smooth muscle cells (VSMCs) into the luminal surface is critical to the anastomotic NH lesion formation.^8,9Several animal models with native or synthetic graft accesses have been used to gain insight into the pathologic mechanisms of NH lesion development.^10,11 However, these studies lacked the critical component of chronic kidney disease (CKD), and whether CKD plays a role in NH lesion formation remains unknown. CKD has been implicated in the development of atherosclerosis along with a host of other deranged factors such as hemodynamic forces, inflammatory mediators, platelet activation, coagulation cascade, and metabolic factors.^12,13 In this study, we used a murine model of CKD modified from Gagnon and Gallimore,¹⁴ to assess the effect of CKD on NH formation after AV fistula creation.     

An Immunohistochemical Analysis of Implanted Woven Dacron and Expanded Polytetrafluoroethylene Grafts in Humans

Abstract: An immunohistochemical analysis was performed to clarify the healing process in implanted vascular grafts in human. Eight woven Dacron grafts and 6 expanded polytetrafluoroethylene grafts were obtained following redo surgery, limb amputation, and autopsy. The implantation periods ranged from 5 days to 148 months. The antibodies used for the analysis were specific to a-actin (smooth muscle cells), macrophages, von Willebrand factor (endothelial cells), fibrin, elastin, collagen types 1–5, CD3 (T cells), and CD20 (B cells). At 5 and 24 days after implantation, thrombi containing red blood cells and fibrin covered the anastomotic lines and some of the luminal surfaces of the grafts. Macrophages were scattered throughout the thrombi. At 11–148 months after implantation, either a single layer of endothelial cells or a thin layer of fibrin covered the anastomotic segments of the grafts, and smooth muscle cells and collagen fibers were seen forming anastomotic intimal hyperplasia (AIH). The AIH in the grafts at 94 and 148 months after implantation was almost the same thickness and length as that in the grafts at 11–36 months after implantation. Apart from the anastomotic segments, a connective tissue matrix containing collagen fibers covered the luminal surfaces, and some thrombi were noted. Most of the collagen present was type 3, in addition to some type 1, 4, and 5 collagen. No type 2 collagen was noted. Some elastin was also detected in the AIH but not in the midportion of the grafts. Some macrophages and T cells were noted in the perigraft tissues.     

The Use of Polytetrafluoroethylene (Gore-Tex) Grafts in Reconstruction of the Urinary Bladder

The safety and histopathologic effects of polytetrafluoroethylene (Gore-Tex) grafts in reconstruction of the urinary bladder were examined. Following partial excision of the bladder Gore-Tex was placed, and the rats were sacrificed at days 7, 14 and 30. Gore-Tex did not cause urine infection, and there was no peritonitis or sepsis in any of the rats. Inflammation around the Gore-Tex diminished after four weeks. Some mononuclear cells and exudate were observed on the inner surface of the Gore-Tex. There was no inflammation or fibrosis in the mucosa and muscular layers of the remaining bladder. In this study the Gore-Tex graft was found to be an infection resistant, urine impermeable material, with no adverse effects on the urinary bladder. Gore-Tex is suggested as a safe material for the reconstruction of the urinary bladder.     

Relationship Between Fibril Length and Tissue Ingrowth in the Healing of Expanded Polytetrafluoroethylene Grafts

Purpose To determine whether fibril length is correlated with graft healing as well as cellular and capillary ingrowth in a canine carotid implantation model.Methods Expanded polytetrafluoroethylene (ePTFE) vascular grafts with three different fibril lengths (30, 60, and 90µm) were implanted into the carotid artery in dogs. They were retrieved 4 weeks later, and subjected to histomorphometric analysis.Results Endothelial healing was best in the 60-µm grafts. Not only cellular ingrowth but also capillary ingrowth was most evident in the 60-µm grafts, followed by the 90-µm grafts and then the 30-µm grafts.Conclusion Better endothelial healing of ePTFE vascular grafts is correlated with more cellular and capillary ingrowth, but more cellular and capillary ingrowth is not correlated with longer fibril length or higher air porosity.     

Activator of G Protein Signaling 3 Promotes Epithelial Cell Proliferation in PKD

The activation of heterotrimeric G protein signaling is a key feature in the pathophysiology of polycystic kidney diseases (PKD). In this study, we report abnormal overexpression of activator of G protein signaling 3 (AGS3), a receptor-independent regulator of heterotrimeric G proteins, in rodents and humans with both autosomal recessive and autosomal dominant PKD. Increased AGS3 expression correlated with kidney size, which is an index of severity of cystic kidney disease. AGS3 expression localized exclusively to distal tubular segments in both normal and cystic kidneys. Short hairpin RNA–induced knockdown of endogenous AGS3 protein significantly reduced proliferation of cystic renal epithelial cells by 26 ± 2% (P < 0.001) compared with vehicle-treated and control short hairpin RNA–expressing epithelial cells. In summary, this study suggests a relationship between aberrantly increased AGS3 expression in renal tubular epithelia affected by PKD and epithelial cell proliferation. AGS3 may play a receptor-independent role to regulate Gα subunit function and control epithelial cell function in PKD.Polycystic kidney disease (PKD) is one of the most common genetic diseases found in humans.¹ The genetic mutation(s) associated with PKD results in fundamental changes in the signal processing of multiple extrinsic cues, leading to abnormal proliferation, fluid secretion, cell polarity and differentiation, and proliferation of the epithelial cells within the kidney and other organs.¹ Heterotrimeric G proteins are key components in the control and integration of the signaling pathways activated in the pathogenesis of fluid-filled cyst or ectatic duct formation in PKD. The traditional activation of intracellular signal transduction pathways after hormonal or mechanical stimulation of target cells was believed to be exclusively through cell surface G protein–coupled receptors (GPCR). In fact, polycystin 1, the major causative cystic protein in autosomal dominant PKD (ADPKD), is considered to behave as a GPCR and regulate heterotrimeric G protein signaling.^2–4 In this report, however, we identified a novel GPCR-independent mechanism to regulate heterotrimeric G protein function in renal epithelial cells through the actions of activator of G protein signaling 3 (AGS3). AGS3 was originally discovered using a yeast-based screening system^5,6 and classified as a group II guanine dissociation inhibitor because of its ability to bind preferentially to inactive Gαi/o subunits complexed with guanine dinucleotide phosphate (GDP) at multiple G protein regulatory or GoLoco motif repeats.⁷ The biologic role of AGS3 has been studied in lower invertebrates and nonrenal mammalian organs; AGS3 regulates the orientation of the mitotic spindle, cAMP production, membrane protein transport, and asymmetric cell division.⁷ These functions have relevance to the pathogenesis of PKD, so we initiated this study to investigate whether AGS3 could be involved in mediating increased proliferation in cystic renal tubular epithelia.Immunoblot analysis demonstrated a marked increase in renal AGS3 protein expression in two distinct murine models of autosomal recessive PKD (ARPKD) compared with their genetic control strains (Figure 1). Increased renal AGS3 protein was detected in the Balb/c polycystic kidney (BPK) mouse at postnatal day 21, which exhibits an advanced stage of cystogenesis, compared with Balb/c mice (Ba; Figure 1A). Similarly, renal AGS3 mRNA (Supplemental Figure 1) and protein (Figure 1B) were observed to increase temporally from weeks 8 to 26 in the polycystic kidney (PCK) rats versus Sprague-Dawley (SD) rats. The low to absent expression of AGS3 in normal rat kidneys is consistent with previously published findings.^8–10 This may be attributed to the exclusive localization to the distal tubular epithelial cells (Figure 2), which compose only a small percentage of the total renal cell population in the kidney. No visible expression was detected in the proximal tubules (Figure 2C), glomeruli (Figure 2C), and blood vessels (Figure 2D). It is interesting to note that the liver, which is the most prevalent extrarenal organ affected by ARPKD, showed increased AGS3 expression with exclusive localization to the biliary epithelial cells (Supplemental Figure 2).Open in a separate windowFigure 1.Increased expression of AGS3 protein in the kidneys from murine models of ARPKD. (A and B) Representative immunoblot analysis for AGS3 and AGS5/LGN expression in mouse (A) and rat kidneys (B). (A) Ba and BPK rat kidney lysates (n = 3 to 4 kidneys per group) at postnatal day 21 (PN21) are examined. (B) SD and PCK rat kidney lysates (n = 4 to 6 rat samples per time point and group) at postnatal weeks 8 (WK8), 16 (WK16), and 26 (WK26) are examined. From these findings, the PCK rat kidneys exhibit a temporal increase in the AGS3 protein between weeks 8 and 26. No change in the expression of AGS5/LGN is determined. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; A) or β-actin (B) is used to ensure equal loading of the protein samples.Open in a separate windowFigure 2.Immunolocalization of AGS3 in ARPKD rat kidneys. AGS3 localization is performed in the kidneys of SD (A, C, and D) and PCK (B, E, and F) rats. Affinity-purified AGS3 antibody (pep32) is incubated at a 1:250 dilution on kidney sections from SD (C and D) and PCK (E and F) rats. As a negative control, SD (A) and PCK (B) rat kidneys are incubated with the primary AGS3 antibody in the presence of the competing AGS3 peptide conjugate or normal rodent serum (data not shown). The brown diaminobenzidine staining demonstrates the specific localization of the AGS3 protein within distinct cell types in the kidney. Sections are counterstained with hematoxylin. Arrowhead in E signifies the magnified view of the area in F. n = 4 kidneys per group.Similar increases in AGS3 protein expression were noted in ADPKD models (Figure 3). The Pkd1^v/v mouse kidney exhibited higher expression of AGS3 compared with the normal C57Bl/6 mouse kidney at postnatal day 14 (Figure 3A). Moreover, robust expression of AGS3 was observed in human ADPKD kidneys (n = 5 kidneys) with minimal, if any, expression detected in normal human kidneys (n = 4 kidneys; Figure 3, B and C). The overexpression of AGS3 in the kidney seemed to be specific to PKD, because other hypertensive rat models with (Dahl S) or without (SHR) renal damage expressed little to no expression of AGS3 protein (Supplemental Figure 3). The expression of AGS5/LGN (Figure 1 and Supplemental Figure 3), a closely related AGS3 homolog (approximately 60% amino acid sequence identity), was found to be unchanged among the various rat kidney groups.Open in a separate windowFigure 3.Increased renal AGS3 expression in a mouse model and humans with ADPKD. (A and B) Representative immunoblot analysis is performed in kidneys obtained from Pkd1^v/v hypomorphic mouse (A) and humans with ADPKD kidneys (B). (A) Normal C57Bl/6 and cystic Pkd1^v/v hypomorph kidneys are harvested at postnatal day 14 and analyzed for AGS3 and AGS5/LGN protein expression. The Pkd1^v/v mouse model of ADPKD exhibits a point mutation in the Pkd1 gene, resulting in inefficient cleavage of polycystin 1 (PC1), which is necessary for optimal activity of PC1.²⁸ (B) Normal human kidneys (n = 4) and human ADPKD kidneys (n = 5) are analyzed for the expression of AGS3. (C) Densitometry of the human AGS3 bands are analyzed (P < 0.001). β-actin is used to determine equal loading of the lanes.To investigate the relationship between AGS3 with epithelial cell proliferation, we generated lentiviral vectors expressing short hairpin RNA (shRNA) molecules targeted to AGS3 and evaluated their efficacy using an AGS3 overexpression system derived from a porcine renal epithelial LLC-PK₁ cell line (Supplemental Figure 4). Using the two most efficient AGS3 shRNA-expressing lentiviral vectors (3 and 4), as determined in Supplemental Figure 4, endogenous AGS3 protein was specifically decreased in only the renal epithelial Ba and BPK cells expressing the combined AGS3 shRNA (denoted as “A” in Figure 4A). The decreased AGS3 protein was associated with a significant reduction (P < 0.001) by 26 ± 2% in the proliferation of the BPK epithelial cells overexpressing the AGS3 shRNA versus the control shRNA. No significant change in either AGS3 protein expression (Figure 4A) or cell proliferation (P > 0.05; Figure 4B) was detected in the control shRNA-expressing cells (denoted as “C”) compared with the vehicle-treated cells (denoted as “V” in Figure 4A).Open in a separate windowFigure 4.AGS3 promotes increased epithelial cell number in a Gβγ-dependent pathway. Ba and BPK epithelial cells are transduced at a multiplicity of infection of 40 with lentiviral vectors expressing shRNA (control or specifically targeted to AGS3) or GRK2ct cDNA. (A through C) For the AGS3 knockdown experiments, the two most effective AGS3 shRNAs (see Supplemental Figure 4) are used for the Western blot (A), cell proliferation assay (B), and cAMP assay (C). (A) Western blot analysis demonstrates a specific reduction in AGS3 expression after combined AGS3 shRNA knockdown (A) compared with control shRNA (C) or vehicle-treated cells (V). No off-target knockdown of AGS5/LGN or β-actin is detected using the AGS3 shRNA. Br, brain (positive control); V, vehicle; A, AGS3 shRNA; C, control (scrambled) shRNA. (B) The genetically modified Ba and BPK cells are aliquotted into 96-well plates and examined for cell number 24 hours later by CyQuant fluorescence assay. *P < 0.001, significant difference between vehicle-treated cells. (C) cAMP levels are examined by ELISA using cell lysates obtained from the genetically modified Ba and BPK cells treated with vehicle or transduced with lentiviral vectors expressing control or specific AGS3 shRNA (n = 6 to 10 samples per group). (D) BPK epithelial cells are incubated with H-89 (10 and 50 μM) for 24 hours before determination of cell numbers by CyQuant fluorescence assay. (E) BPK epithelial cells are incubated with or without Rp-adenosine-3′,5′-monophosphorothioate (20 μM), a specific PKA inhibitor, for 5 hours. Cells are collected for protein isolation to determine the expression of AGS3 by Western blot analysis. Densitometry is performed to determine the band intensities. (F) BPK epithelial cells genetically modified to overexpress GRK2ct are aliquotted into a 96-well plate; 24 hours later, the cell proliferation is measured using the CyQuant fluorescence assay and compared with vehicle-treated BPK cells. *P < 0.001, significant difference between vehicle-treated cells. Numbers of samples in each group are shown in each graph.The mechanism(s) by which AGS3 regulates heterotrimeric G protein activity in the renal epithelia has yet to be elucidated. AGS3 competes with free Gβγ subunits for binding to Gαi/o-GDP subunits, and this would provide the opportunity for AGS3 to regulate downstream signal transduction pathways by either inhibiting Gαi- or activating Gβγ-dependent pathways. Gαi subunits directly inhibit adenylyl cyclase (AC) activity.¹¹ Elevated levels of cAMP are a common observance in many animal models of PKD,¹² and several currently active clinical trials are based on reducing cyst formation through the targeting of AC-dependent pathways.¹² Thus, we initially postulated that AGS3 in renal epithelial cells may play an important role in regulating the activation of cAMP/PKA pathways. In our study, however, we did not observe a relationship between endogenous AGS3 expression with cAMP production (Figure 4C). Reductions in the levels of AGS3 did not result in any difference in cAMP levels in either the presence (data not shown) or the absence (Figure 4C) of a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine. The inability of AGS3 to regulate cAMP production is consistent with a previous study by Sato et al.¹³ that used transfected cell lines overexpressing AGS3. Conversely, there is evidence of the activation of cAMP/PKA pathways in neuronal cells as a result of AGS3-mediated effects.^14,15 After drug withdrawal, AGS3 was found to potentiate the activity of AC and cAMP production as a result of a PKA-dependent increase of AGS3 expression.¹⁴ In our study, we measured a dosage-dependent reduction in cell number after inhibition with a specific PKA inhibitor (H-89; Figure 4D), which suggests that basal epithelial cell activity of cAMP/PKA pathways can mediate proliferation. Unlike the findings in the neuronal cells,¹⁴ we did not observe an increase in AGS3 protein expression after inhibition with another specific PKA antagonist, Rp-adenosine-3′,5′-monophosphorothioate (Figure 4E). The lack of a regulatory effect by AGS3 on the cAMP/PKA pathway may be attributed to low basal AC activity in the renal epithelia using serum-free cell culture conditions. It is likely that additional stimuli, which are normally available in the mammalian circulation, may be required to prime the renal epithelial cells to uncover the AGS3-dependent role on cAMP/PKA similar to the previous neuronal cell studies.^14,15Instead of altering cAMP production, we examined whether the sequestration of Gαi subunits by AGS3 prevented the re-association with free Gβγ dimers to activate Gβγ-mediated downstream signaling cascades, as observed in previous behavior modification^15,16 and mammalian brain development studies.¹⁷ To block the activity of Gβγ dimers in renal epithelia, the C-terminal region of the bovine GPCR kinase 2 (GRK2ct) was overexpressed in the BPK renal epithelia using lentiviral vectors. The GRK2ct contains a pleckstrin homology domain that binds free Gβγ subunits to regulate its signaling activity.¹⁸ The cell proliferation in the GRK2ct-expressing cells was significantly reduced (P < 0.001) by 68 ± 1% compared with the vehicle-treated BPK cells (Figure 4F). The AGS3 mediated Gβγ-dependent mechanism to promote cell proliferation has not been well defined, but there may be direct interactions with AC to promote cAMP production,¹⁴ mitogen-activated protein kinase pathways¹⁹ or possibly through the regulation of mitotic spindle orientation.¹⁷ Sanada et al.¹⁷ showed that either reductions in endogenous AGS3 or overexpression of GRK2ct in neural progenitor cells shifted the axis of cell cleavage from a horizontal to vertical plane. From this study, the orientation of the progenitor cell divisions was correlated with cell fate (i.e., there was increased neuronal fate with a concomitant loss of the progenitor cell pool).¹⁷ At the present time, there is an emerging albeit controversial role for the regulation of the mitotic spindle in PKD. Dysregulation of the mitotic spindle in renal epithelial cells is believed to be associated with promoting aberrant epithelial cell proliferation, tubular dilation, and cyst formation.^20–23 On a subcellular level, AGS3 was found to co-localize with Gαi3 at the plasma membrane and mitotic spindle poles (Supplemental Figure 5), which could suggest a potential role for AGS3 in mitotic spindle pole orientation. Our findings in conjunction with previously published studies in other organs and invertebrate systems suggest that there is a unique role for AGS3 to regulate proliferative signaling cascades through a Gβγ-dependent manner.In summary, our study provides the first evidence demonstrating aberrant expression of AGS3, a novel receptor-independent regulator of heterotrimeric G protein, in the kidneys from multiple mammalian models of ARPKD and ADPKD. Our data suggest a novel mechanism by which AGS3-Gα signaling cassettes may be involved in activating noncanonical G protein pathways to promote renal epithelial cell proliferation. AGS3 may play a fundamentally important role in the integration of multiple converging signaling pathways to transition normal renal tubular epithelia toward a pathologically cystic disease phenotype. Our study may open the door to new investigations into the development of anticystic drugs to target AGS3-Gα complexes to treat PKD and possibly other proliferative diseases.     

Phosphorylcholine Coating of ePTFE Grafts Reduces Neointimal Hyperplasia in Canine Model

2 versus 0.53 ± 0.13 mm², respectively; p= 0.008). Furthermore, the BrdU labeling index in the graft neointimal tissues was significantly smaller (p < 0.001) in the treated group (2.64 ± 0.77%) as compared with the control group (5.07 ± 0.83%). These data demonstrate that PC coating of ePTFE significantly reduces graft neointimal hyperplasia and cell proliferation in a canine carotid artery bypass model. The application of PC within the ePTFE graft effectively blocks tissue ingrowth from the adjacent native vessel, thereby preserving the anastomosis luminal diameter.     

Measurement of Vascular Input Impedance in Infrainguinal Vein Grafts

n = 40) or claudication (n= 17) were prospectively studied. At the time of revascularization, simultaneously acquired intraluminal pressure and blood flow waveforms were digitized at 200 Hz and subjected to Fourier transformation in near real-time. Input impedance was calculated at baseline (immediately after unclamping) and after stimulation with either papaverine or completion arteriography. Resistance (R _in) was calculated as mean pressure divided by mean blood flow (Q). Characteristic impedance (Z ₀) was calculated as the mean of harmonics 3–10. Intraoperative data acquisition required approximately 5 min, utilized the completion angiography cannula already in place, and was uncomplicated in all patients. Stimulation with either papaverine or arteriography resulted in increased Q (72 ± 7 to 146 ± 11 ml/min, p < 0.001), decreased R _in (126 ± 13 to 52 ± 4 × 10³ dyne · s · cm⁻⁵, p < 0.001), and slightly decreased Z ₀ (18 ± 2 to 15 ± 1, p = 0.002). After a mean follow-up of 20 months, the 2-year primary patency, secondary patency, limb salvage, and survival rates were 61 ± 8%, 74 ± 7%, 76 ± 6%, and 86 ± 6%, respectively. Primary patency was not associated with any of the clinical variables studied including age, sex, smoking history, history of previous vascular surgery, hypertension, coronary artery disease, diabetes mellitus, creatinine, indication for revascularization (claudication versus limb salvage), anesthesia (general versus regional), or level of distal anastomosis (popliteal versus infrapopliteal). Furthermore, there was no association between primary patency and baseline Q, baseline R _in, or stimulated Z ₀. However, using univariate analysis, patency was positively associated with decreased stimulated R _in (p= 0.002), elevated stimulated Q (p= 0.006), and decreased baseline Z ₀ (p= 0.02). Multiple regression analysis identified stimulated R _in as the only independent predictor of primary patency (p= 0.002). Stimulated R _in≥ 50 × 10³ dyne · s · cm⁻⁵ was 71% sensitive and 65% specific for graft failure. It is concluded that 1) vascular input impedance can be simply and reliably measured in the operating room, and 2) elevated stimulated R _in is an independent predictor of primary patency.     

pcDNA3.1/hTIMP-1-EGFP真核表达载体的构建及其在血管平滑肌细胞中的表达

目的 构建标记有增强型绿色荧光蛋白(EGFP)的人基质金属蛋白酶组织抑制因子-1(hTIMP-1)真核表达质粒pcDNA3.1/hTIMP-1-EGFP,并通过转染大鼠血管平滑肌细胞(SMCs)验证hTIMP-1的表达.方法 将hTIMP-1及编码EGFP的互补脱氧核糖核酸(cDNA)序列通过扩增、酶切、插入pcDNA3.1质粒的多克隆位点,构建pcDNA3.1/hTIMP-1-EGFP真核表达质粒;应用脂质体介导的基因转染技术,将基因导入SMCs(pcDNA3.1/hTIMP-1-EGFP转染组).应用荧光显微镜观察EGFP的表达情况;流式细胞仪检测转染效率;逆转录-聚合酶链反应(RT-PCR)、蛋白免疫印迹法(Western blotting)等方法检测hTIMP-1的表达情况;明胶酶谱法检测基质金属蛋白酶MMP-2、MMP-9活性.用空质粒pcDNA3.1转染SMCs(空质粒pcDNA3.1转染组)和未转染质粒的SMCs(未转染组)作为对照.结果 pcDNA3.1/hTIMP-1-EGFP转染组重组质粒转染SMCs后,SMCs生长受到抑制;转染24 h后在荧光显微镜下可见强绿色荧光,流式细胞仪检测转染效率约为15%;RT-PCR检测结果显示,SMCs出现646 bp目的基因;Western blotting检测显示,在转染后的血管SMCs中有hTIMP-1表达;明胶酶谱法检测结果显示,MMP-2、MMP-9活性降低;与空质粒pcDNA3.1转染组和未转染组比较差异有统计学意义(P＜0.05). 结论 TIMP-1真核表达质粒的构建和在血管SMCs中的表达,为TIMP-1基因治疗的研究奠定了基础.