Cardiovascular disease is one of the leading contributors to morbidity and mortality in ESRD patients (1). Current therapies have failed to reduce this vascular risk, with renin-angiotensin system blockade (2), BP control (3), and 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibition (4) all failing to show benefits in clinical trials.
One of the major contributors to vascular disease in ESRD is endothelial injury because uremia is associated with dysfunction (5) and even loss (6) of the endothelium. Early-outgrowth endothelial progenitor-like cells (EPLCs), a novel bone-marrow-derived mononuclear cell population, promote endothelial repair and new endothelial growth (7) through the release of soluble factors that activate local angiogenic processes (8,9), maintaining healthy blood vessels and preserving tissue perfusion in the setting of ischemic injury (10,11). Importantly, preliminary data suggest that uremia induces early-outgrowth EPLC dysfunction, at least in vitro (12).
Nocturnal hemodialysis (NHD) greatly augments uremic toxin clearance compared with conventional thrice weekly hemodialysis (CHD) through a combination of increased dialysis duration and frequency. NHD is associated with better cardiovascular outcomes, including improved endothelial function (13), attenuated vascular calcification (14), reduced BP (15), and regression of left ventricular hypertrophy (16) when compared with its conventional counterpart. Intriguingly, conversion to NHD has also been associated with symptomatic and structural improvements in severe cases of ischemic vascular disease (17). Recently, we demonstrated that NHD is associated with improved circulating early-outgrowth EPLC number and in vitro function (18). Here we show for the first time that (1) early-outgrowth EPLCs derived from CHD patients are unable to restore perfusion in ischemic tissue in vivo, and (2) NHD is associated with a reversal of this early-outgrowth EPLC angiogenic defect when tested in an in vivo rodent model of ischemic vascular disease.
Study Population and Methods
This protocol was approved by the Research Ethics Boards of the Toronto General Hospital, University Health Network, and St. Michael's Hospital. Two groups of age- and gender-matched ESRD patients were studied: (1) CHD patients (n = 10), and (2) NHD patients (n = 9). A separate group of healthy individuals (n = 5) was also studied as controls. CHD and NHD patients had been on their respective dialysis modalities for a minimum duration of 6 months. None of the patients had any acute illness or symptomatic cardiovascular disease (including congestive heart failure and acute coronary syndrome). Written informed consent was obtained from each patient.
Clinical Data Collection
In both ESRD cohorts, clinical assessment (including weight, height, BP, and routine dialysis-related biochemical analyses) was performed. Seated BP in each patient after 5 minutes of rest was measured during a clinic visit. Blood samples were obtained at midweek and predialysis for CHD patients. To minimize circadian variation and to replicate steady-state CHD and NHD conditions, blood samples were drawn at the same time of day for NHD patients (a minimum of 4 hours after the end of a regular NHD session). Dialysis dose per treatment was estimated by equilibrated Kt/V (eKt/V) as described by Daugirdas and colleagues (19), where eKt/V = spKt/V − 0.6(spKt/V)/t + 0.03, where spKt/V is the single-pool Kt/V, K is the delivered clearance, t is the dialysis time, and V is the urea distribution volume. Single-pool Kt/V was determined using the blood urea reduction ratio (20). Cardiovascular medications were documented, including diuretics, β-blockers, angiotensin-converting enzyme inhibitors, angiotensin receptor antagonists, calcium channel blockers, and HMG-CoA reductase inhibitors. The dose of erythropoiesis-stimulating agent prescribed was also documented because of its well established effects on early-outgrowth EPLC number and function (21).
NHD patients received hemodialysis at home for 6 to 8 hours, 5 to 6 nights per week. Vascular access was achieved through an internal jugular catheter (Uldall Catheter, Cook Critical Care, Bloomington, IN) or an arteriovenous fistula. A dialysate flow rate of 350 ml/min, a blood flow rate of 200 to 300 ml/min, and Polyflux polyamide dialyzers (Gambro, Lund, Sweden or Baxter, McGaw Park, IL) were used for each treatment. CHD patients received hemodialysis for 4 hours three times per week via similar vascular access. A blood flow rate of 400 ml/min, a dialysate flow rate of 500 to 750 ml/min, and F80 polysulfone dialyzers (Fresenius Medical Care, Lexington, MA) were used for CHD delivery. Unfractionated heparin was used for anticoagulation on CHD and NHD.
Early-Outgrowth EPLC Isolation
Early-outgrowth EPLCs were isolated by enriched medium isolation as described previously (18,22). Briefly, peripheral venous blood was collected from ESRD patients and healthy control subjects. The mononuclear cell fraction was isolated by Ficoll–Paque density gradient (Becton Dickinson, Mississauga, Canada) centrifugation and washed 3 times with PBS (Sigma-Aldrich, Mississauga, Canada), and cells were plated at a density of 106 mononuclear cells/cm2 on fibronectin-coated culture slides (Becton Dickinson) in endothelial cell basal medium-2 (Lonza) supplemented with endothelial growth medium SingleQuots and 20% fetal bovine serum. Cells were trypsinized after 10 days of culture and washed with PBS. Cells (5 × 105) were resuspended in 1 ml of PBS and then used for in vivo injection.
Unilateral Chronic Hindlimb Ischemia Model, Tissue Preparation, and Histology
All animal studies were approved by the St. Michael's Hospital Animal Ethics Committee in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication no 85-23, revised 1996). Male athymic nude rats (6 to 8 weeks, Charles River, Montreal, Canada) were obtained and maintained at the St. Michael's Hospital Animal Research Vivarium in a temperature-controlled (22°C) room with ad libitum access to commercial irradiated standard rat chow and autoclaved water. Rats underwent unilateral left-sided chronic hindlimb ischemia induction as described previously (23). Briefly, rats were anesthetized with inhaled isoflurane (2% to 3%). Using aseptic technique, the left common iliac artery and small proximal branches were exposed and doubly ligated with 4-0 suture. The incision was closed in layers, and the animals were recovered. One day after left common iliac artery ligation, athymic nude rats were injected with saline or 5 × 105 early-outgrowth EPLCs at five intramuscular sites using a 1-cc syringe and 25-gauge needle in the ischemic hindlimb, as described previously (24), using aseptic technique and under inhaled isoflurane (2% to 3%) anesthetic. The total volume of injection per rat was 1 cc (0.2 cc per site). At study end 27 days later, rats were sacrificed, and the right (nonischemic) and left (ischemic) hindlimb muscles were immediately isolated and immersion-fixed in 10% neutral-buffered formalin, embedded in cryostat matrix (Tissue-Tek, Sakura, Kobe, Japan), or flash frozen in liquid nitrogen. Formalin-fixed tissues were routinely processed, embedded in paraffin, and sectioned (5 μm thickness). Endothelial cell density within formalin-fixed, paraffin-embedded sections of ischemic hindlimb tissue was assessed using staining with biotin-conjugated isolectin GS-B4 (Invitrogen, Burlington, Canada, catalog number I21414), and the Vectastain ABC system (Vector Laboratories, Burlington, Canada) was used for detection of specific isolectin B4 staining as described previously (25). Omission of isolectin B4 served as the negative control. Quantification of ten nonoverlapping 160× fields was performed for each animal in a blinded fashion with a grid technique.
RNA Extraction and Quantitative Real-Time PCR
Quantitative real-time PCR (RT-PCR) was performed using SYBR green. In brief, frozen rat hindlimb tissue, stored at −80°C, was homogenized (Polytron, Kinematica GmbH, Littau, Switzerland), and total RNA was isolated using TRIzol reagent (Life Technologies, Grand Island, NY). Total RNA (4 μg) was treated with RQ1 DNAse (1 U/μl) (Promega, Madison, WI) to remove genomic DNA. RNA was reverse transcribed into cDNA with 1 μl of random hexamers (2 μg/μl), 2.5 μl of 10 mmol/L dNTP mix, 0.5 μl RNase inhibitor (40 U/μl) (Roche, Indianapolis, IN), and 0.5 μl avian myeloblastosis virus reverse transcriptase (25 U/μl) (Roche). cDNA samples were stored at −20°C until further analysis. RT-PCR was performed on an ABI Prism 7900HT Fast PCR System (Applied Biosystems, Foster City, CA). Sequence-specific primers were designed to span exon-exon boundaries using Primer Express software version 1.5 (Applied Biosystems) and were obtained from Sigma-Aldrich. Primer sequences are provided in supplemental Table 1. Experiments were performed in triplicate, and data analysis was performed using the Applied Biosystems Comparative CT method.
All values were referenced to the mRNA transcript levels of the housekeeper gene Rpl13a (10) and are presented as ratios of mRNA levels between the ischemic (left) and nonischemic (right) limbs indexed to the value found for saline-treated animal controls.
Contrast-Enhanced Ultrasound Imaging
Contrast-enhanced ultrasound imaging of the proximal hindlimb adductor muscles was performed with gated pulse inversion imaging (HDI 5000, Philips Ultrasound, Andover, MA) at a mechanical index of 1.0 and a transmission frequency of 3.3 MHz (L7-4 transducer). Perfusion in the adductor muscles was assessed during continuous intravenous infusion of lipid microbubbles (1 × 107 min−1) as described previously (23).
All data are shown as mean ± SEM unless otherwise stated. Differences between groups were analyzed by one-way ANOVA with post hoc Fisher's least significant difference or Mann–Whitney test. Bivariate correlation analysis was performed with Spearman's rho test. All statistics were performed using GraphPad Prism 4.00 for Windows (GraphPad Software, San Diego, CA) or SPSS 15.0 for Windows (SPSS, Chicago, IL). A change was considered statistically significant if P < 0.05.
Demographic and Clinical Characteristics
Five healthy control individuals, 10 prevalent CHD, and 9 prevalent NHD patients were enrolled. The NHD and CHD patients were age and gender matched. Relevant demographic data are presented in Table 1, and clinical data are presented in Table 2. All patients were nonsmokers. NHD was associated with an increased sessional dialysis dose (eKt/V, P < 0.05) and reduced predialysis urea and phosphate (P < 0.05 for both). NHD was also associated with higher hemoglobin concentrations despite similar erythropoiesis-stimulating agent usage (P < 0.05). Although systolic and diastolic BP were not significantly different between the CHD and NHD cohorts, CHD patients used a greater number of antihypertensives to achieve this level of control. HMG-CoA reductase usage was slightly higher in the CHD cohort.
NHD Is Associated with an Improved Ability of Early-Outgrowth EPLCs to Restore Microvascular Perfusion in Ischemic Tissue
The effect of dialysis modality on human early-outgrowth EPLC-mediated neovascularization ability was assessed in a rodent model of chronic unilateral ischemic vascular disease. One day after ligation of the left common iliac artery, athymic nude rats were injected at five intramuscular sites in the ischemic leg with saline or 5 × 105 early-outgrowth EPLCs derived from ex vivo culture of patient-derived mononuclear cells. Twenty-eight days postligation, contrast-enhanced ultrasound demonstrated that injection of NHD patient–derived early-outgrowth EPLCs induced a robust increase in microvascular perfusion of the ischemic limb similar in magnitude to that achieved with healthy control-derived cell injection (Figure 1). In contrast, CHD patient–derived cell injection had no significant effect on microvascular perfusion when compared with saline-injected controls (Figure 1 and Supplemental Movies). Similar trends were seen in microvascular blood volume, an index of blood volume contained within the ischemic hindlimb microvasculature (Figure 1). Dialysis dose, as measured by eKt/V, correlated positively with microvascular blood flow (r = 0.536, P < 0.05) and volume (r = 0.656, P < 0.01) (Figure 2).
NHD Is Associated with an Improved Ability of Early-Outgrowth EPLCs to Promote Neovascularization in Ischemic Tissue
To assess the structural correlates of the observed changes in microvascular perfusion, sectioning of ischemic hindlimb tissue and lectin staining were performed to investigate differences in capillary density. Correlating with the improvement in microvascular perfusion and blood volume, NHD and healthy control early-outgrowth EPLC treatment were associated with significant and equivalent increases in capillary density when compared with saline-treated controls (Figure 3). In contrast, CHD cell treatment did not alter capillary density compared with saline-treated controls (Figure 3). As observed with microvascular perfusion parameters, dialysis dose correlated positively with capillary density (r = 0.713, P < 0.01) (Figure 4).
Injected NHD Patient-Derived Early-Outgrowth EPLCs Promote the Expression of Endogenous Angiopoietin 1
To assess the molecular mechanisms underlying NHD early-outgrowth EPLC-mediated improvements in capillary density and microvascular perfusion, the expression of key angiogenic factors was interrogated by quantitative RT-PCR 4 weeks after cell therapy. Although endogenous expression of the vessel-stabilizing factor angiopoietin 1 (Ang1) in the ischemic hindlimb of CHD early-outgrowth EPLC-treated animals was no different from saline-treated controls, rat Ang1 mRNA levels were markedly upregulated in NHD cell-treated ischemic hindlimb tissue (saline: 1.00 ± 0.36 arbitrary units [AU], CHD: 0.86 ± 0.51 AU, NHD: 5.48 ± 2.68 AU, P < 0.05). In contrast, expression of the early angiogenic factors vascular endothelial growth factor (VEGF-A), angiopoietin 2 (Ang2), and stromal-cell-derived factor-1α (SDF-1α) was not significantly increased above saline-treated controls in the CHD or NHD early-outgrowth EPLC-treated ischemic hindlimbs (supplemental Table 2).
Vascular disease is common in ESRD patients and is a substantial contributor to the poor outcomes seen in uremia (26,27). Importantly, no evidence-based therapies exist to treat vascular disease in ESRD, with most studies reporting a poor prognosis regardless of treatment choice (4,28,29). In the study presented here, we show that (1) CHD is associated with a marked inability of early-outgrowth EPLCs to restore blood flow and microvasculature in an in vivo model of ischemic vascular disease, and (2) NHD is associated with significant restoration of early-outgrowth EPLC function in this model.
Multiple lines of evidence support the concept that early-outgrowth EPLCs participate in endothelial repair. Preclinical proof-of-concept studies have demonstrated that early-outgrowth EPLCs promote neovascularization in ischemic tissue (30–32) and inhibit capillary rarefaction in chronic kidney disease (10). Clinical studies have demonstrated that human early-outgrowth EPLC administration induces significant proangiogenic benefits on top of standard-of-care therapy in the peripheral vascular disease (33) and postmyocardial infarction (11,34) settings. Strong epidemiologic links have also been made between circulating early-outgrowth EPLC number and cardiovascular risk (35,36).
Endothelial dysfunction plays a key role in the pathogenesis of ESRD-associated vascular complications (37). Interestingly, the ESRD milieu appears to adversely affect mechanisms of endothelial repair critical in maintaining endothelial homeostasis in the face of injury. Previous reports have demonstrated that early-outgrowth EPLCs of predialysis chronic kidney disease (38) and dialysis (12) patients demonstrate dysfunction in vitro. These reports tested the ability of early-outgrowth EPLCs to migrate along an in vitro VEGF-A chemotactic gradient and to incorporate into “vascular networks” with human umbilical vein endothelial cells in culture (12,39). As our understanding of early-outgrowth EPLC biology has evolved, it has become clear that the proangiogenic functions of these cells are complex, and, in contrast to true endothelial progenitor cells, are likely not due to transdifferentiation into progeny endothelial cells and incorporation into existing vasculature (40). Instead, early-outgrowth EPLCs play an important supportive role in the neovascularization response, likely recruiting local angiogenic mechanisms (9,41,42) and promoting tissue protection (10). Recognizing that the exact mechanisms by which early-outgrowth EPLCs mediate their proangiogenic effects are still incompletely understood, we chose instead in this translational study to examine the effects of hemodialytic modality on the function of these cells using a model of ischemic vascular disease that integrates these mechanisms in vivo. In doing so, we demonstrate for the first time that early-outgrowth EPLCs derived from CHD patients demonstrate a marked defect in their ability to restore perfusion and microvasculature in ischemic leg tissue, whereas augmenting uremic clearance with NHD can return the in vivo angiogenic capacity of these cells to near-normal levels.
An interesting observation from our study is the demonstration that injected human early-outgrowth EPLCs lead to the endogenous production of Ang1 in ischemic hindlimb muscle. In line with the complexity of the angiogenic process, multiple signaling molecules play stage-specific roles in coordinating new blood vessel growth (43). Ischemic hindlimb muscle was collected 28 days postligation, at a time when the levels of factors such as VEGF-A, Ang2, and SDF-1α that are activated early in angiogenesis have returned back to baseline (23). In contrast, Ang1 expression is often maintained for weeks after ischemia onset (23). Ang1 plays a critical role in the maturation of newly formed vascular networks in postnatal angiogenesis through the recruitment of pericytes (44) and has been shown to promote neovascularization in the setting of ischemia (45). Because exogenously administered early-outgrowth EPLCs have been shown to regulate host angiogenesis (41), the finding that NHD patient–derived cells, but not CHD patient–derived cells, upregulated endogenous Ang1 expression and ischemic neovascularization raises important mechanistic questions beyond the scope of the study presented here. Because NHD could alter many aspects of early-outgrowth EPLC biology, future studies are needed to clarify the relevance of this finding.
In an effort to assess the effect of hemodialysis regimen on early-outgrowth EPLC angiogenicity in vivo, the study presented here compared early-outgrowth EPLCs derived from age-, gender-, and comorbidity-matched CHD and NHD patients with those derived from healthy controls. Although it is possible that unmeasured variables might explain some of the differences observed in early-outgrowth EPLC angiogenicity, CHD and NHD patients were equally matched for known variables affecting early-outgrowth EPLC function, including age, smoking status, and diabetes prevalence. Although CHD and NHD patients did differ in antihypertensive usage, this is a known improvement associated with NHD (13,15) and may reflect improvements in endothelial function (13). Importantly, although early-outgrowth EPLCs from CHD patients had similar effects as saline injection, cells derived from NHD patients exerted angiogenic activity similar to cells derived from younger healthy controls, suggesting dramatic differences in function that seem unlikely to be fully explained by nondialytic factors.
In conclusion, this study is the first to document that NHD is associated with significant improvements in the in vivo angiogenic activity of peripheral-blood-derived early-outgrowth EPLCs using an immunodeficient rodent model of ischemic vascular disease. The study presented here suggests that augmenting dialysis dose might restore the ability of early-outgrowth EPLCs to mediate vascular repair. Future studies are urgently needed to test the effects of NHD on early-outgrowth EPLC angiogenicity in the setting of human vascular disease.
Part of this material was presented at the annual meeting of the American Society of Nephrology; November 16 through 21, 2010; Denver, CO, and was sponsored by a Grant-in-Aid from the Heart and Stroke Foundation of Ontario (C.T.C., H.L.P, D.Y, NA 6605), and a Resident Research Grant from The Physicians' Services, Inc., Foundation (D.Y., R08-57). Dr. Darren Yuen is supported by a KRESCENT postdoctoral fellowship. Dr. Christopher Chan is the R. Fraser Elliott Chair in Home Dialysis at the University Health Network. Dr. Howard Leong-Poi is supported by a Phase II Clinician Scientist Award from the Heart and Stroke Foundation of Ontario and an Early Researcher Award from the Ministry of Research and Innovation, Ontario, Canada. We thank Dr. R. Gilbert and Dr. A. Advani for their helpful comments in the preparation of the manuscript, Ms. K. Thai and Ms. S. Advani for their technical assistance, and Ms. M. Mitchell and Mr. N. Cheung for their expert technical assistance. H.L.-P. and C.T.C. contributed equally to this work.
Published online ahead of print. Publication date available at www.cjasn.org.
Supplemental information for this article is available online at www.cjasn.org.
1. U.S. Renal Data System: 2009 Annual Data Report: Atlas of Chronic Kidney Disease and End-Stage Renal Disease in the United States, Bethesda, MD, National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Disease, 2009
2. Zannad F, Kessler M, Lehert P, Grunfeld JP, Thuilliez C, Leizorovicz A, Lechat P: Prevention of cardiovascular events in end-stage renal disease: Results of a randomized trial of fosinopril and implications for future studies. Kidney Int 70: 1318–1324, 2006
3. Li Z, Lacson E Jr, Lowrie EG, Ofsthun NJ, Kuhlmann MK, Lazarus JM, Levin NW: The epidemiology of systolic blood pressure and death risk in hemodialysis patients. Am J Kidney Dis 48: 606–615, 2006
4. Fellstrom BC, Jardine AG, Schmieder RE, Holdaas H, Bannister K, Beutler J, Chae DW, Chevaile A, Cobbe SM, Gronhagen-Riska C, De Lima JJ, Lins R, Mayer G, McMahon AW, Parving HH, Remuzzi G, Samuelsson O, Sonkodi S, Sci D, Suleymanlar G, Tsakiris D, Tesar V, Todorov V, Wiecek A, Wuthrich RP, Gottlow M, Johnsson E, Zannad F: Rosuvastatin and cardiovascular events in patients undergoing hemodialysis. N Engl J Med 360: 1395–1407, 2009
5. Stam F, van Guldener C, Schalkwijk CG, ter Wee PM, Donker AJ, Stehouwer CD: Impaired renal function is associated with markers of endothelial dysfunction and increased inflammatory activity. Nephrol Dial Transplant 18: 892–898, 2003
6. Amann K, Breitbach M, Ritz E, Mall G: Myocyte/capillary mismatch in the heart of uremic patients. J Am Soc Nephrol 9: 1018–1022, 1998
7. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM: Isolation of putative progenitor endothelial cells for angiogenesis. Science 275: 964–967, 1997
8. Sieveking DP, Buckle A, Celermajer DS, Ng MK: Strikingly different angiogenic properties of endothelial progenitor cell subpopulations: Insights from a novel human angiogenesis assay. J Am Coll Cardiol 51: 660–668, 2008
9. Urbich C, Aicher A, Heeschen C, Dernbach E, Hofmann WK, Zeiher AM, Dimmeler S: Soluble factors released by endothelial progenitor cells promote migration of endothelial cells and cardiac resident progenitor cells. J Mol Cell Cardiol 39: 733–742, 2005
10. Yuen DA, Connelly KA, Advani A, Liao C, Kuliszewski MA, Trogadis J, Thai K, Advani SL, Zhang Y, Kelly DJ, Leong-Poi H, Keating A, Marsden PA, Stewart DJ, Gilbert RE: Culture-modified bone marrow cells attenuate cardiac and renal injury in a chronic kidney disease rat model via a novel antifibrotic mechanism. PLoS ONE 5: e9543, 2010
11. Assmus B, Honold J, Schachinger V, Britten MB, Fischer-Rasokat U, Lehmann R, Teupe C, Pistorius K, Martin H, Abolmaali ND, Tonn T, Dimmeler S, Zeiher AM: Transcoronary transplantation of progenitor cells after myocardial infarction. N Engl J Med 355: 1222–1232, 2006
12. Choi JH, Kim KL, Huh W, Kim B, Byun J, Suh W, Sung J, Jeon ES, Oh HY, Kim DK: Decreased number and impaired angiogenic function of endothelial progenitor cells in patients with chronic renal failure. Arterioscler Thromb Vasc Biol 24: 1246–1252, 2004
13. Chan CT, Harvey PJ, Picton P, Pierratos A, Miller JA, Floras JS: Short-term blood pressure, noradrenergic, and vascular effects of nocturnal home hemodialysis. Hypertension 42: 925–931, 2003
14. Yuen D, Pierratos A, Richardson RM, Chan CT: The natural history of coronary calcification progression in a cohort of nocturnal haemodialysis patients. Nephrol Dial Transplant 21: 1407–1412, 2006
15. Culleton BF, Walsh M, Klarenbach SW, Mortis G, Scott-Douglas N, Quinn RR, Tonelli M, Donnelly S, Friedrich MG, Kumar A, Mahallati H, Hemmelgarn BR, Manns BJ: Effect of frequent nocturnal hemodialysis vs conventional hemodialysis on left ventricular mass and quality of life: A randomized controlled trial. JAMA 298: 1291–1299, 2007
16. Chan CT, Floras JS, Miller JA, Richardson RM, Pierratos A: Regression of left ventricular hypertrophy after conversion to nocturnal hemodialysis. Kidney Int 61: 2235–2239, 2002
17. Chan CT, Mardirossian S, Faratro R, Richardson RM: Improvement in lower-extremity peripheral arterial disease by nocturnal hemodialysis. Am J Kidney Dis 41: 225–229, 2003
18. Chan CT, Li SH, Verma S: Nocturnal hemodialysis is associated with restoration of impaired endothelial progenitor cell biology in end-stage renal disease. Am J Physiol Renal Physiol 289: F679–F684, 2005
19. Daugirdas JT, Greene T, Depner TA, Gotch FA, Star RA: Relationship between apparent (single-pool) and true (double-pool) urea distribution volume. Kidney Int 56: 1928–1933, 1999
20. Jindal KK, Manuel A, Goldstein MB: Percent reduction in blood urea concentration during hemodialysis (PRU). A simple and accurate method to estimate Kt/V urea. ASAIO Trans 33: 286–288, 1987
21. Heeschen C, Aicher A, Lehmann R, Fichtlscherer S, Vasa M, Urbich C, Mildner-Rihm C, Martin H, Zeiher AM, Dimmeler S: Erythropoietin is a potent physiologic stimulus for endothelial progenitor cell mobilization. Blood 102: 1340–1346, 2003
22. Ormiston ML, Deng Y, Stewart DJ, Courtman DW: Innate immunity in the therapeutic actions of endothelial progenitor cells in pulmonary hypertension. Am J Respir Cell Mol Biol 43: 546–554, 2010
23. Leong-Poi H, Kuliszewski MA, Lekas M, Sibbald M, Teichert-Kuliszewska K, Klibanov AL, Stewart DJ, Lindner JR: Therapeutic arteriogenesis by ultrasound-mediated VEGF165 plasmid gene delivery to chronically ischemic skeletal muscle. Circ Res 101: 295–303, 2007
24. Takeshita S, Isshiki T, Ochiai M, Eto K, Mori H, Tanaka E, Umetani K, Sato T: Endothelium-dependent relaxation of collateral microvessels after intramuscular gene transfer of vascular endothelial growth factor in a rat model of hindlimb ischemia. Circulation 98: 1261–1263, 1998
25. Walter DH, Rittig K, Bahlmann FH, Kirchmair R, Silver M, Murayama T, Nishimura H, Losordo DW, Asahara T, Isner JM: Statin therapy accelerates reendothelialization: A novel effect involving mobilization and incorporation of bone marrow-derived endothelial progenitor cells. Circulation 105: 3017–3024, 2002
26. O'Hare AM, Feinglass J, Sidawy AN, Bacchetti P, Rodriguez RA, Daley J, Khuri S, Henderson WG, Johansen KL: Impact of renal insufficiency on short-term morbidity and mortality after lower extremity revascularization: Data from the Department of Veterans Affairs' National Surgical Quality Improvement Program. J Am Soc Nephrol 14: 1287–1295, 2003
27. Foley RN, Parfrey PS, Sarnak MJ: Clinical epidemiology of cardiovascular disease in chronic renal disease. Am J Kidney Dis 32: S112–S119, 1998
28. Wanner C, Krane V, Marz W, Olschewski M, Mann JF, Ruf G, Ritz E: Atorvastatin in patients with type 2 diabetes mellitus undergoing hemodialysis. N Engl J Med 353: 238–248, 2005
29. O'Hare A, Johansen K: Lower-extremity peripheral arterial disease among patients with end-stage renal disease. J Am Soc Nephrol 12: 2838–2847, 2001
30. Kalka C, Masuda H, Takahashi T, Kalka-Moll WM, Silver M, Kearney M, Li T, Isner JM, Asahara T: Transplantation of ex vivo
expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci U S A 97: 3422–3427, 2000
31. Kocher AA, Schuster MD, Szabolcs MJ, Takuma S, Burkhoff D, Wang J, Homma S, Edwards NM, Itescu S: Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med 7: 430–436, 2001
32. Murohara T, Ikeda H, Duan J, Shintani S, Sasaki K, Eguchi H, Onitsuka I, Matsui K, Imaizumi T: Transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization. J Clin Invest 105: 1527–1536, 2000
33. Tateishi-Yuyama E, Matsubara H, Murohara T, Ikeda U, Shintani S, Masaki H, Amano K, Kishimoto Y, Yoshimoto K, Akashi H, Shimada K, Iwasaka T, Imaizumi T: Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: A pilot study and a randomised controlled trial. Lancet 360: 427–435, 2002
34. Schachinger V, Erbs S, Elsasser A, Haberbosch W, Hambrecht R, Holschermann H, Yu J, Corti R, Mathey DG, Hamm CW, Suselbeck T, Assmus B, Tonn T, Dimmeler S, Zeiher AM: Intracoronary bone marrow-derived progenitor cells in acute myocardial infarction. N Engl J Med 355: 1210–1221, 2006
35. Vasa M, Fichtlscherer S, Aicher A, Adler K, Urbich C, Martin H, Zeiher AM, Dimmeler S: Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ Res 89: E1–E7, 2001
36. Hill JM, Zalos G, Halcox JP, Schenke WH, Waclawiw MA, Quyyumi AA, Finkel T: Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med 348: 593–600, 2003
37. Guerin AP, Pannier B, Metivier F, Marchais SJ, London GM: Assessment and significance of arterial stiffness in patients with chronic kidney disease. Curr Opin Nephrol Hypertens 17: 635–641, 2008
38. de Groot K, Bahlmann FH, Sowa J, Koenig J, Menne J, Haller H, Fliser D: Uremia causes endothelial progenitor cell deficiency. Kidney Int 66: 641–646, 2004
39. Herbrig K, Pistrosch F, Oelschlaegel U, Wichmann G, Wagner A, Foerster S, Richter S, Gross P, Passauer J: Increased total number but impaired migratory activity and adhesion of endothelial progenitor cells in patients on long-term hemodialysis. Am J Kidney Dis 44: 840–849, 2004
40. Hirschi KK, Ingram DA, Yoder MC: Assessing identity, phenotype, and fate of endothelial progenitor cells. Arterioscler Thromb Vasc Biol 28: 1584–1595, 2008
41. Popa ER, Harmsen MC, Tio RA, van der Strate BW, Brouwer LA, Schipper M, Koerts J, De Jongste MJ, Hazenberg A, Hendriks M, van Luyn MJ: Circulating CD34+ progenitor cells modulate host angiogenesis and inflammation in vivo
. J Mol Cell Cardiol 41: 86–96, 2006
42. Rehman J, Li J, Orschell CM, March KL: Peripheral blood “endothelial progenitor cells” are derived from monocyte/macrophages and secrete angiogenic growth factors. Circulation 107: 1164–1169, 2003
43. Carmeliet P: Mechanisms of angiogenesis and arteriogenesis. Nat Med 6: 389–395, 2000
44. Asahara T, Chen D, Takahashi T, Fujikawa K, Kearney M, Magner M, Yancopoulos GD, Isner JM: Tie2 receptor ligands, angiopoietin-1 and angiopoietin-2, modulate VEGF-induced postnatal neovascularization. Circ Res 83: 233–240, 1998
45. Saif J, Schwarz TM, Chau DY, Henstock J, Sami P, Leicht SF, Hermann PC, Alcala S, Mulero F, Shakesheff KM, Heeschen C, Aicher A: Combination of injectable multiple growth factor-releasing scaffolds and cell therapy as an advanced modality to enhance tissue neovascularization. Arterioscler Thromb Vasc Biol 30: 1897–1904, 2010