eNOS transfection of adipose-derived stem cells yields bioactive nitric oxide production and improved results in vascular tissue engineering.

This study evaluates the durability of a novel tissue engineered blood vessel (TEBV) created by seeding a natural vascular tissue scaffold (decellularized human saphenous vein allograft) with autologous adipose-derived stem cells (ASC) differentiated into endothelial-like cells. Previous work with this model revealed the graft to be thrombogenic, likely due to inadequate endothelial differentiation as evidenced by minimal production of nitric oxide (NO). To evaluate the importance of NO expression by the seeded cells, we created TEBV using autologous ASC transfected with the endothelial nitric oxide synthase (eNOS) gene to produce NO. We found that transfected ASC produced NO at levels similar to endothelial cell (EC) controls in vitro which was capable of causing vasorelaxation of aortic specimens ex vivo. TEBV (n = 5) created with NO-producing ASC and implanted as interposition grafts within the aorta of rabbits remained patent for two months and demonstrated a non-thrombogenic surface compared to unseeded controls (n = 5). Despite the xenograft nature of the scaffold, the TEBV structure remained well preserved in seeded grafts. In sum, this study demonstrates that upregulation of NO expression within adult stem cells differentiated towards an endothelial-like lineage imparts a non-thrombogenic phenotype and highlights the importance of NO production by cells to be used as endothelial cell substitutes in vascular tissue engineering applications.

Endothelial differentiation of adipose-derived stem cells from elderly patients with cardiovascular disease.

Adipose-derived stem cells (ASCs) possess significant therapeutic potential for tissue engineering and regeneration. This study investigates the endothelial differentiation and functional capacity of ASCs isolated from elderly patients. Isolation of ASCs from 53 patients (50-89 years) revealed that advanced age or comorbidity did not negatively impact stem cell harvest; rather, higher numbers were observed in older donors (>70 years) than in younger. ASCs cultured in endothelial growth medium-2 for up to 3 weeks formed cords upon Matrigel and demonstrated acetylated-low-density lipoprotein and lectin uptake. Further stimulation with vascular endothelial growth factor and shear stress upregulated endothelial cell-specific markers (CD31, von Willebrand factor, endothelial nitric oxide synthase, and VE-cadherin). Inhibition of the PI(3)K but not mitogen-activated protein kinase pathway blocked the observed endothelial differentiation. Shear stress promoted an anti-thrombogenic phenotype as demonstrated by production of tissue-plasminogen activator and nitric oxide, and inhibition of plasminogen activator inhibitor-1. Shear stress augmented integrin α(5)ß(1) expression and subsequently increased attachment of differentiated ASCs to basement membrane components. Finally, ASCs seeded onto a decellularized vein graft resisted detachment despite application of shear force up to 9 dynes. These results suggest that (1) advanced age and comorbidity do not negatively impact isolation of ASCs, and (2) these stem cells retain significant capacity to acquire key endothelial cell traits throughout life. As such, adipose tissue is a practical source of autologous stem cells for vascular tissue engineering.

The role of hypoxia in stem cell differentiation and therapeutics.

Stem cells differentiate into a variety of cell lines, making them attractive for tissue engineering and regenerative medicine. Specific microenvironmental cues regulate self-renewal and differentiation capabilities. Oxygen is an important component of the cellular microenvironment, serving as both metabolic substrate and signaling molecule. Oxygen has been shown to have a variety of effects on embryonic and adult stem cells. This review examines the role of hypoxia in regulating stem cell biology, specifically focusing on growth, maintenance of pluripotency, differentiation, and production of growth factors. Particular attention is paid to hypoxia and stem cells in relation to therapeutic angiogenesis. We conclude that further study is needed to optimize the use of hypoxia as a stimulus for various stem cell functions, including its potential role in therapeutic angiogenesis.

Differentiation of adult stem cells into smooth muscle for vascular tissue engineering.

BACKGROUND: Herein we evaluate the potential of adipose-derived stem cells (ASC) to differentiate into smooth muscle cells (SMC) and their potential for use in a tissue-engineered vascular graft. MATERIALS AND METHODS: We isolated ASC (CD13+29+90+) from the peri-umbilical adipose tissue of patients undergoing vascular surgery, and cultured them in media containing angiotensin II (AngII), sphingosylphosphorylcholine (SPC), or transforming growth factor-beta 1 (TGFß1) for up to 3 weeks. SMC differentiation was assessed by (1) expression of early (calponin, caldesmon) and late (myosin heavy chain, MHC) SMC markers by RT-PCR, qPCR and Western blot, and (2) contraction upon plating on collagen gel. Differentiated ASCs were seeded onto a vascular graft (decellularized saphenous vein) within a bioreactor, and cell attachment was determined using confocal microscopy. RESULTS: Prior to differentiation, ASC expressed low levels of all three molecular markers. After culture in each differentiating medium, the extent of up-regulation of calponin, caldesmon, and MHC was variable across all cell lines. After seeding onto collagen gel, ASCs differentiated in SPC and TGFß1 exhibit contractile properties, similar to smooth muscle cell controls. Differentiated stem cells adhered and proliferated on the vascular graft. CONCLUSION: These data suggest that human adipose-derived stem cells (1) exhibit variable expression of SMC molecular markers after differentiation, (2) exhibit a contractile phenotype after differentiation with SPC and TGFß1, and (3) proliferate on a vascular graft scaffold. Thus, ASCs are potentially useful in the construction of autologous arteries.

Availability of adipose-derived stem cells in patients undergoing vascular surgical procedures.

BACKGROUND: Most research evaluating adipose-derived stem cells (ASC) uses tissue obtained from young, healthy patients undergoing plastic surgical procedures. Given the propensity of other adult stem cell lines to diminish with increasing patient age and co-morbidities, we assess the availability of ASC in elderly patients undergoing vascular surgical procedures, and evaluate their acquisition of endothelial cell (EC) traits to define their potential use in vascular tissue engineering. METHODS AND METHODS: Adipose tissue obtained by liposuction from patients undergoing vascular procedures (n = 50) was digested with collagenase and centrifuged to remove mature adipocytes. The resultant number of cells, defined as the stromal-vascular (SV) pellet, was quantified. Following a 7-d culture period and negative selection for CD31 and CD45, the resultant number of ASC was quantified. After culture in differentiating media (EMG-2), ASCs were tested for the acquisition of endothelial-specific traits (expression of CD31, realignment in shear, cord formation on Matrigel). RESULTS: The SV pellet contained 2.87 ± 0.34 × 10(5) cells/g fat, and the resultant number of ASCs obtained was 1.41 ± 0.18 × 10(5) cells/g fat. Flow cytometry revealed a homogeneous ASC population (>98% positive for CD13, 29, 90). Advanced age or co-morbidity (obesity, diabetes, renal or peripheral vascular disease) did not significantly alter yield of ASC. After culture in differentiating media (EMG-2), ASCs acquired each of the endothelial-specific traits. CONCLUSION: ASC isolation appears independent of age and co-morbidities, and ASCs harvested from patients with vascular disease retain their ability to differentiate into endothelial-like cells. Adipose tissue, therefore, is a practical source of autologous, adult stem cells for vascular tissue engineering.

Linear shear conditioning improves vascular graft retention of adipose-derived stem cells by upregulation of the alpha5beta1 integrin.

Use of adult adipose-derived stem cells (ASCs) as endothelial cell substitutes in vascular tissue engineering is attractive because of their availability. However, when seeded onto decellularized vascular scaffolding and exposed to physiological fluid shear force, ASCs are physically separated from the graft lumen. Herein we have investigated methods of increasing initial ASC attachment using luminal precoats and a novel protocol for the gradual introduction of shear stress to optimize ASC retention. Fibronectin coating of the graft lumen increased ASC attachment by nearly sixfold compared with negative controls. Gradual introduction of near physiological fluid shear stress using a novel bioreactor whereby flow rate was increased every second at a rate of 1.5 dynes/cm(2) per day resulted in complete luminal coverage compared with near complete cell loss following conventional daily abrupt increases. An upregulation of the alpha(5)beta(1) integrin was evinced following exposure to shear stress, which accounts for the observed increase in ASC retention on the graft lumen. These results indicated a novel method for seeding, conditioning, and retaining of adult stem cells on a decellularized vein scaffold within a high-shear stress microenvironment.

Endothelial differentiation of adipose-derived stem cells: effects of endothelial cell growth supplement and shear force.

BACKGROUND: Adipose tissue is a readily available source of multipotent adult stem cells for use in tissue engineering/regenerative medicine. Various growth factors have been used to stimulate acquisition of endothelial characteristics by adipose-derived stem cells (ASC). Herein we study the effects of endothelial cell growth supplement (ECGS) and physiological shear force on the differentiation of ASC into endothelial cells. MATERIALS AND METHODS: Human ASC (CD13(+)29(+)90(+)31(-)45(-)) were isolated from periumbilical fat, cultured in ECGS media (for up to 3 wk), and exposed to physiological shear force (12 dynes for up to 8 d) in vitro. Endothelial phenotype was defined by cord formation on Matrigel, acetylated-low density lipoprotein (acLDL) uptake, and expression of nitric oxide synthase (eNOS), von Willebrand factor (vWF), and CD31 (platelet endothelial cell adhesion molecule, PECAM). Additionally, cell thrombogenicity was evaluated by seeding canine autologous ASC onto vascular grafts implanted within the canine arterial circulation for 2 wk. RESULTS: We found that undifferentiated ASC did not display any of the noted endothelial characteristics. After culture in ECGS, ASC formed cords in Matrigel but failed to take up acLDL or express the molecular markers. Subsequent exposure to shear resulted in stem cell realignment, acLDL uptake, and expression of CD31; eNOS and vWF expression was still not observed. Grafts seeded with cells grown in ECGS (+/- shear) remained patent (six of seven) at 2 wk but had a thin coat of fibrin along the luminal surfaces. CONCLUSIONS: This study suggests that (1) ECGS and shear promote the expression of several endothelial characteristics in human adipose-derived stem cells, but not eNOS or vWF; (2) their combined effects appear synergistic; and (3) stem cells differentiated in ECGS appear mildly thrombogenic in vitro, possibly related, in part, to insufficient eNOS expression. Thus, while the acquisition of several endothelial characteristics by adult stem cells derived from adipose tissue suggests these cells are a viable source of autologous cells for cardiovascular regeneration, further stimulation/modifications are necessary prior to using them as a true endothelial cell replacement.

Have endovascular procedures negatively impacted general surgery training?

OBJECTIVE: Technological advances in vascular surgery have changed the field dramatically over the past 10 years. Herein, we evaluate the impact of endovascular procedures on general surgery training. METHODS: National operative data from the Residency Review Committee for Surgery were examined from 1997 through 2006. Total major vascular operations, traditional open vascular operations and endovascular procedures were evaluated for mean number of cases per graduating chief general surgery resident (GSR) and vascular surgery fellow (VSF). RESULTS: As endovascular surgical therapies became widespread, GSR vascular case volume decreased 34% over 10 years, but VSF total cases increased 78%. GSR experience in open vascular operations decreased significantly, as evidenced by a 52% decrease (P < 0.0001) in elective open AAA repair. VSFs have also seen significant decreases in open vascular procedures. Experience in endovascular procedures has increased for both general surgery and vascular residents, but the increase has been much larger in absolute number for VSFs. CONCLUSIONS: GSR experience in open vascular procedures has significantly decreased as technology has advanced within the field. Unlike VSFs, this loss has not been replaced by direct experience with endovascular training. These data demonstrate the impact technology can have on how we currently train general surgeons. New educational paradigms may be necessary in which either vascular surgery as an essential component is abandoned or training in catheter-based interventions becomes required.

Development of a tissue-engineered bypass graft seeded with stem cells.

The gold standard conduit for bypass of diseased small-diameter arteries remains autologous vascular tissue. In the absence of such tissue, patients are offered bypass with prosthetic material, with far less durable results. Vascular tissue engineering, the creation of a vascular conduit by seeding a tubular scaffold with various cells, may offer an alternative approach to this difficult situation. Herein we review some of the significant challenges that remain in designing an ideal vascular conduit and outline potential solutions offered by a graft created by seeding natural vascular tissue (decellularized vein allograft) with readily available autologous cells (adipose-derived stem cells).

Inflammatory and angiogenic mRNA levels are altered in a supraspinatus tendon overuse animal model.

Shoulder overuse injuries, especially those to the supraspinatus tendon of the rotator cuff, are common musculoskeletal disorders. Unfortunately, little is known about the disease etiology and pathogenesis. The objective of this study was to determine the levels of specific inflammatory and angiogenic markers in a rat supraspinatus tendon overuse injury model. We hypothesized that inflammation would not be present early in the overuse protocol. Conversely, we hypothesized that the overuse protocol would result in increased angiogenesis early. Increases in five-lipoxygenase activating protein, cyclooxygenase-2, vascular endothelial growth factor, and von Willebrand factor were evaluated by use of reverse transcription-polymerase chain reaction from 1 day through 16 weeks of treadmill running (overuse protocol). These results provide important information on the role of angiogenesis and inflammation in the disease process. Future studies will further evaluate the mechanisms of the disease process as well as potential targeted treatment modalities.