Prospective purification of perivascular presumptive mesenchymal stem cells from human adipose tissue: process optimization and cell population metrics across a large cohort of diverse demographics.

BACKGROUND: Adipose tissue is an attractive source of mesenchymal stem cells (MSC) as it is largely dispensable and readily accessible through minimally invasive procedures such as liposuction. Until recently MSC could only be isolated in a process involving ex-vivo culture and their in-vivo identity, location and frequency remained elusive. We have documented that pericytes (CD45-, CD146+, and CD34-) and adventitial cells (CD45-, CD146-, CD34+) (collectively termed perivascular stem cells or PSC) represent native ancestors of the MSC, and can be prospectively purified using fluorescence activated cell sorting (FACS). In this study we describe an optimized protocol that aims to deliver pure, viable and consistent yields of PSC from adipose tissue. We analysed the frequency of PSC within adipose tissue, and the effect of patient and procedure based variables on this yield. METHODS: Within this twin centre study we analysed the adipose tissue of n = 131 donors using flow cytometry to determine the frequency of PSC and correlate this with demographic and processing data such as age, sex, BMI and cold storage time of the tissue. RESULTS: The mean number of stromal vascular fraction (SVF) cells from 100 ml of lipoaspirate was 34.4 million. Within the SVF, mean cell viability was 83 %, with 31.6 % of cells being haematopoietic (CD45+). Adventitial cells and pericytes represented 33.0 % and 8 % of SVF cells respectively. Therefore, a 200 ml lipoaspirate would theoretically yield 23.2 million viable prospectively purified PSC - sufficient for many reconstructive and regenerative applications. Minimal changes were observed in respect to age, sex and BMI suggesting universal potential application. CONCLUSIONS: Adipose tissue contains two anatomically and phenotypically discreet populations of MSC precursors - adventitial cells and pericytes - together referred to as perivascular stem cells (PSC). More than 9 million PSC per 100 ml of lipoaspirate can be rapidly purified to homogeneity using flow cytometry in clinically relevant numbers potentially circumventing the need for purification and expansion by culture prior to clinical use. The number and viability of PSC are minimally affected by patient age, sex, BMI or the storage time of the tissue, but the quality and consistency of yield can be significantly influenced by procedure based variables.

Adipose stromal cells-secreted neuroprotective media against neuronal apoptosis.

Transplantation of pluripotent adipose stem/stromal cells (ASC) alleviates tissue damage and improves functional deficits in both stroke and cardiovascular disease animal models. Recent studies indicate that the primary mechanism of ASC-induced repair may not be directly related to tissue regeneration through differentiation, but rather through paracrine mechanisms provided by secreted pro-survival and repair-inducing trophic factors. In this study, we have found that ASC-conditioned medium (ASC-CM) potently protected cerebellar granule neurons (CGN) from apoptosis induced by serum and potassium deprivation. Neural cell protection was mostly attributable to activated caspase-3 and Akt-mediated neuroprotective pathway signaling. Specific neutralization of neurotrophic factor activity demonstrated that serum and potassium deprivation-induced Akt-mediated neuroprotection and caspase-3-dependent apoptosis were mainly modulated by IGF-1. These data suggest that of the many neuroprotective factors secreted by ASC, IGF-1 is the major factor that mediates protection against serum and potassium deprivation-induced CGN apoptosis. This study establishes a mechanistic basis supporting the therapeutic application of ASC for neurological disorders, specifically through paracrine support provided by trophic factor secretion.

Adipose stromal cells-conditional medium protected glutamate-induced CGNs neuronal death by BDNF.

The delivery of factors secreted by adipose stromal cells (ASC) to the brain may be a viable neuroprotective therapeutic option. In this study, we investigated the bioactivity of ASC-conditioned medium (ASC-CM) in glutamate-induced neurotoxicity and found that the ASC-CM significantly blocked glutamate neurotoxicity. We identified the brain-derived neurotrophic factor (BDNF) in the ASC-CM by using Western blot and demonstrated that this activity was critical for the neuroprotective effect of ASC-CM in excytotoxicity models. Furthermore, inactivating BDNF also attenuated the suppression by ASC-CM of glutamate-induced caspase-3 activity, but not p38 phosphorylation. These findings suggest that among ASC secrete a potent combination of factors, BDNF play a major role in neuroprotection against excytotoxicity.

[Adipose tissue stromal cells -- multipotent cells with therapeutic potential for stimulation of angiogenesis in tissue ischemia].

Results of studies of properties of adipose tissue stromal cells are summarized in this review. It contains data on separation and cultivation of these cells as well as description of their antigenic characteristics, ability to differentiate into cells of mesenchymal and nonmesenchymal origin, angiogenic potential, and potential for transfection and transfer of therapeutic genes. Analysis of perspectives of clinical use of adipose tissue stromal cells in therapy of cardiovascular and other diseases is also presented.

[Adipose stromal cells--plastic type of cells with high therapeutic potential].

Much effort has been made in searching for multipotent cell types with high therapeutic potentials for repair of damaged tissue. Through enzymatic digestion of fat tissue, it is possible to obtain a large number of stromal cells. Isolated cells show a high proliferate capacity in culture. All this makes adipose stromal cells (ASC) promising candidates for their use in cell therapy. This review is focused on analyzing the surface antigen profile of isolated population of ASC, expression of angiogenic factors by these cells, as well as on their differentiation potential. A high percentage of ASC population initially express the progenitor cell marker CD34, but during culturing, cells exhibit a mesenchymal cell phenotype and express CD29, CD105, CD106, CD166. Culturing ASC in specific differentiation media induces expression of early markers of differentiated mesenchymal cells, such as adipocytes, chondrocytes and osteoblasts, as well as myoblasts, cardiomyocytes and neural cells. It has been also shown that ASC have a strong pro-angiogenic potential, they are able to secret growth factors, such as VEGF, HGF, bFGF and others, which stimulate survival and proliferation of endothelial cells. In addition, systemic or local delivery of ASC to mice with hindlimb ischemia stimulates recovery of injured tissue and blood flow. Potential clinical uses of ASCs are discussed in the review.

Advantages of short-lived positron-emitting radioisotopes for intracoronary radiation therapy with liquid-filled balloons to prevent restenosis.

UNLABELLED: Balloon catheters filled with liquid radioisotopes provide excellent dose homogeneity for intracoronary radiation therapy but are associated with risk for rupture or leakage. We hypothesized that the safety of liquid-filled balloons may be improved once positron emitters with half-lives below 2 h are used instead of the high-energy beta-emitters 166Ho, 186Re, or 188Re, all of which have a longer half-life of at least 17 h. METHODS: To support this concept, the suitability of 18F (half-life, 109.8 min), 68Ga (half-life, 67.6 min), 11C (half-life, 20.4 min), 13N (half-life, 9.97 min), and 15O (half-life, 2.04 min) for intracoronary radiation therapy was evaluated. Potential tissue penetration of positron radiation was assessed in a series of phantom experiments using Gafchromic film. Antiproliferative efficacy of positrons emitted by 68Ga was investigated in vitro using cultured bovine aortic smooth muscle cells (BASMCs), and was compared with gamma-radiation emitted by 137Cs. To characterize the remaining risk, we estimated radiotoxicity after accidental intravascular balloon rupture on the basis of tabulated isotope-specific doses (ICRP 53) and compared these values with 188Re. RESULTS: Half-dose depth of tissue penetration measured in phantom experiments was 0.29 mm for 18F, 0.42 mm for 11C, 0.54 mm for 13N, 0.79 mm for 15O, and 0.9 mm for 68Ga. Irradiation of cultured BASMCs with positron radiation (68Ga) induced dose-dependent inhibition of proliferation with complete proliferative arrest at doses exceeding 6 Gy. ED(50) and ED(80) were 2.5 +/- 0.4 Gy (mean +/- SD) and 4.4 +/- 0.8 Gy, respectively. Antiproliferative efficacy was equal to that of the 662-keV gamma-radiation emitted by 137Cs (ED(50), 3.8 +/- 0.2 Gy; ED(80), 8.0 +/- 0.3 Gy). Estimates made for patient whole-body and organ doses were generally below 50 mSv/1.85 GBq for all investigated positron emitters. The same dose estimates for 188Re were 6-20 fold higher. CONCLUSION: Among the studied radioisotopes, 68Ga is the most attractive source for liquid-filled balloons because of its convenient half-life, sufficient positron energy (2.92 MeV), documented antiproliferative efficacy, and uncomplicated availability from a radioisotope generator. The safety profile for 68Ga is significantly better than that of 188Re, which suggests this radioisotope should be evaluated further in preclinical studies.

Improved laser-assisted vascular tissue fusion using light-activated surgical adhesive in a porcine model.

Newly developed light-activated surgical adhesives have been investigated as a substitute to traditional protein solders for vascular tissue fusion without the need for sutures. Porcine carotid arteries (n = 6) and femoral arteries (n = 6) were exposed, and an incision was made in the arterial walls using a 16G needle. The surgical adhesive, composed of a poly(L-lactic-co-glycolic acid) scaffold doped with the traditional protein solder mix of porcine serum albumin and indocyanine green dye, was used to close the incisions in conjunction with an 805 nm diode laser. Blood flow was restored to the vessels immediately after the procedure and the incision sites were checked for patency. The new adhesives were flexible enough to be wrapped around the vessels while their solid nature avoided the problems associated with "runaway" of the less viscous liquid protein solders widely used by researchers. The strength and hemostatic abilities of the new surgical adhesives were evaluated in the context of arterial pressure, persistence of hemostasis and presence of any inflammatory reaction after 3 days. After this evaluation period, the surgical procedure was repeated on the carotid arteries (n = 6) and femoral arteries (n = 6) of three additional animals that had been heparinized prior to surgery to closer approximate the conditions seen in a typical vascular surgical setting. The patency rate of both the unheparinized and heparinized vessels was 100% at 3 days post-operative with evidence of intraluminal thrombosis seen in only one of the repaired vessels. The adhesive technique also compared favorably with a previous study conducted using conventional suture techniques. Repairs formed with the adhesive technique were achieved more rapidly than suturing, and acute leakage was observed less frequently. Finally, the adhesive technique produced better histology than the suture technique, suggesting that it has great promise as an alternative to suturing. These initial results indicate that laser-assisted vascular repair using the new adhesives is safe, easy to perform, and contrary to conventional suturing, provides an immediate leak-free closure. In addition, the flexible and moldable nature of the new adhesives allows them to be tailored to a wide range of tissue geometries, thus greatly improving the clinical applicability of laser-assisted tissue repair.

Liquid-filled balloon brachytherapy using (68)Ga is effective and safe because of the short 68-minute half-life: results of a feasibility study in the porcine coronary overstretch model.

BACKGROUND: Liquid-filled balloons for coronary brachytherapy provide significant advantages over solid sources in dose homogeneity but carry the risk of life-threatening radiointoxication after balloon rupture and laboratory contamination in case of a spill. We hypothesized that the positron emitter (68)Ga, with a half-life of only 68 minutes, was well suited to overcome these safety obstacles while providing full therapeutic efficacy. METHODS AND RESULTS: The feasibility, efficacy, and safety of (68)Ga liquid-filled balloon brachytherapy were investigated in the porcine coronary overstretch model. Four groups of 5 balloon-induced coronary lesions were irradiated with 8, 12, 16, and 24 Gy targeted to the adventitia. Ten unirradiated lesions served as controls. Segments treated with 16 or 24 Gy exhibited marked suppression of neointimal proliferation at 28-day follow-up, with quantitative parameters of intraluminal proliferation reduced to <20%. This beneficial effect was not compromised by untoward edge effects. Uninjured but irradiated vessels did not show histological signs of radiation damage. The (68)Ga whole-body dose due to balloon rupture was estimated to be 5 rem/50 mCi treatment activity and compared favorably with that of (188)Re (78 rem/50 mCi). CONCLUSIONS: (68)Ga positron radiation suppresses neointimal proliferation at doses of 16 and 24 Gy. This biological efficacy, coupled with the attractive safety profile, suggests the selection of (68)Ga as an attractive isotope for liquid-filled balloon brachytherapy.

Intrapericardial paclitaxel delivery inhibits neointimal proliferation and promotes arterial enlargement after porcine coronary overstretch.

BACKGROUND: Catheter-based intrapericardial (IPC) delivery of therapeutic agents has recently been demonstrated. Paclitaxel is known to inhibit vascular smooth muscle cell proliferation. This study examined the effect of IPC instillation of paclitaxel on neointimal proliferation after balloon overstretch of porcine coronary arteries. METHODS AND RESULTS: Overstretch injury of coronary arteries was followed by IPC administration of micellar paclitaxel at low dose (LD, 10 mg; n=6) or high dose (HD, 50 mg; n=7) or of control micelles (50 mg, n=5). Animals were euthanized 28 days after balloon dilation. Arterial injury indices were no different among the groups. The neointimal area, maximal intimal thickness, and adventitial thickness were significantly reduced in both LD (0.47+/-0.04 mm(2), 0.43+/-0.03 mm, and 0.35+/-0.02 mm, respectively) and HD (0.51+/-0.06 mm(2), 0.42+/-0.03 mm, and 0. 38+/-0.03 mm, respectively) paclitaxel groups compared with the control group (0.79+/-0.07 mm(2), 0.56+/-0.02 mm, and 0.47+/-0.02 mm, respectively; P:<0.001). Meanwhile, the vessel circumference measured at the external elastic lamina of paclitaxel-treated vessels was significantly larger than the control circumference. Apoptotic cells were found in the neointima. The apoptotic cell percentage was not different between the control (1.72%) and LD (2. 31%) groups but was higher in the HD group (7.07%, P:<0.0001 versus control and LD groups). Immunostaining for matrix metalloproteinase-2 revealed concurrent reduction in the HD group compared with the control and LD groups. CONCLUSIONS: IPC space delivery of a single dose of paclitaxel significantly reduces vessel narrowing in this balloon-overstretch model. This effect is mediated by reduction of neointimal mass as well as positive vascular remodeling.

Differences in the effects of HMG-CoA reductase inhibitors on proliferation and viability of smooth muscle cells in culture.

We investigated the influence of lovastatin, simvastatin and pravastatin on proliferation and viability of vascular smooth muscle cells (SMC) in vitro and studied the effects of lovastatin on a mouse SMC line transgenic for a temperature-sensitive mutant of SV40 large T antigen (TAg), known to inhibit the function of p53 and pRb family members. We found that lovastatin and simvastatin inhibited cell proliferation by provoking G0/G1 phase arrest with concomitant depression of the proliferation antigen Ki-67/MIB-1. Lovastatin at high concentrations of 20 micromol/l caused cell death in the presence of serum but not under serum starved conditions, which was verified on the basis of increased DNA strand breaks, decreased DNA content and morphological alterations seen by electron microscopy. Cell death was also found for simvastatin, whereas pravastatin did not exhibit antiproliferative or cytotoxic effects. Mouse SMC transgenic for TAg did not show any impaired sensitivity to the antiproliferative and cell death inducing effect of lovastatin, but both effects could be antagonized by the supplementation of mevalonate. The data indicate that antiproliferative and cytotoxic effects of lovastatin are caused by the using up of products of mevalonate metabolism and do not require the presence of p53 or pRb.

Studies of renal injury III: lipid-induced nephropathy in type II diabetes.

UNLABELLED: Studies of renal injury III: Lipid-induced nephropathy in type II diabetes. BACKGROUND: Nephrotoxicity from elevated circulating lipids occurs in experimental and clinical situations. We tested the hypothesis that lipid-induced nephropathy causes advanced renal failure in rats with type II diabetes and dyslipidemia. METHODS: First generation (F1) hybrid rats derived from the spontaneous hypertensive heart failure rat (SHHF/Gmi-fa) and the LA/NIH-corpulent rat (LA/N-fa) were studied for 41 weeks while being on specific diets. Group 1 (14 rats) ingested 11.5% protein, 47.9% fat, and 40.6% carbohydrate. Group 2 (8 rats) ingested 26.9% protein, 16.7% animal fat, and 56.4% carbohydrate, and group 3 (20 rats) ingested 20.2% protein, 40.4% soy and coconut oil, and 39.4% carbohydrate. RESULTS: Hyperglycemia was more severe in rat groups 1 and 2 than in group 3. In contrast, circulating cholesterol and hydroperoxide levels were highest in group 3, intermediate in group 2, and lowest in group 1. Group 3 had severe renal failure secondary to glomerulosclerosis and tubulointerstitial disease, with striking deposition of the lipid peroxidation stress biomarker 4-hydroxynonenal in glomeruli and renal microvessels. Moreover, in group 3, increased arterial wall thickness also connoted vascular injury. In contrast, the glycoxidation stress biomarkers pentosidine and carboxymethyl-lysine were preferentially localized to renal tubules of hyperglycemic rats in groups 1 and 2 and did not segregate with the most severe renal injury. Glomerular and interstitial fibrosis was accompanied by proportional increases in renal transforming growth factor-beta1 levels, which were threefold higher in the hypercholesterolemic rats of group 3 than in the hyperglycemic rats of group 1. CONCLUSIONS: Acquisition of non-nodular glomerular sclerosis and tubulointerstitial disease is dependent on lipoxidation stress in rats with type II diabetes. On the other hand, in the absence of hypercholesterolemia, prolonged glycoxidation stress does not appear to be uniquely nephrotoxic.

Hyperplasia in multiple smooth muscle tissues in transgenic mice expressing a temperature-sensitive SV40 T-antigen under the control of smooth muscle alpha-actin regulatory sequences.

Control of smooth muscle cell (SMC) proliferation is of fundamental importance in the development and pathology of the vasculature. To derive vascular SMC with conditional inactivation of negative cell cycle regulatory proteins in the context of smooth muscle protein expression, a 3.4 kb fragment of the mouse SMC alpha-actin promoter was used to target a temperature-sensitive mutant SV40 T antigen (tsA58) to smooth muscle in transgenic mice. Mice with this genotype display a heritable phenotype of abnormal SMC proliferation in the central tail artery, vasa deferentia, seminal vesicles, prostate, and uterus, with the latter resembling uterine leiomyomatosis and prostatic hypertrophy. Neither the aorta nor other viscera manifested abnormal proliferation. Cultures from aorta, vas deferens, seminal vesicle, and kidney tissue were characterized with regard to protein expression, stability, and matrix remodelling capacity. The alpha-actin content/cell was up to 3-4-fold higher, as well as more stable than in primary SMC cultures, suggesting successful selection for propagation of cells expressing this differentiation marker. All cells displayed enhanced growth at the permissive temperature. As an initial functional assessment, the cells were compared to non-transformed mouse aortic SMC with respect to the ability to remodel collagen gel matrices, and demonstrated conservation of this physiologic function. This in vivo analysis of the SMC alpha-actin promoter supports a broader range of smooth muscle-directed expression activity than previously recognized, and establishes the feasibility of its use to direct transgene expression to vascular as well as genito-urinary smooth muscle. The targeted expression of the tsA58 T antigen has yielded transgenic animals with several manifestations of smooth muscle hyperplasia; these animals have in turn permitted the derivation of several murine SMC lines with phenotypic stability and conditionally-modulated proliferation. These cells will allow expansion of derivative transfected smooth muscle cell lines under permissive conditions, as well as oncogene inactivation at the restrictive temperature when desired for functional studies.

NGF activates similar intracellular signaling pathways in vascular smooth muscle cells as PDGF-BB but elicits different biological responses.

The signaling pathways that regulate smooth muscle cell migration and proliferation are incompletely understood. Smooth muscle cells express at least 3 families of receptor tyrosine kinases that mediate cell migration: platelet-derived growth factor (PDGF) receptors, the trk family of neurotrophin receptors, and insulin-like growth factor 1 receptor. The neurotrophin, nerve growth factor (NGF), and insulin-like growth factor 1 induce the migration but not the proliferation of smooth muscle cells, whereas PDGF-BB stimulates both responses. To determine whether distinct signaling pathways downstream of receptor tyrosine kinases specifically mediate smooth muscle cell migration or proliferation, the ligand-induced activation of different signaling pathways in smooth muscle cells was examined. NGF induces prolonged activation of the Shc/MAP kinase pathway and phospholipase Cgamma compared with PDGF-BB. The activation of phosphatidylinositol-3 kinase, however, was 10-fold greater in response to PDGF-BB compared with NGF. Insulin-like growth factor 1 activates only phosphatidylinositol-3 kinase. Pharmacological inhibitors of phosphatidylinositol-3 kinase, Wortmannin and LY294002, inhibit PDGF-BB and NGF-induced migration, whereas an inhibitor of MAP kinase kinase, PD98059, has no effect. Our results suggest that (1) different receptor tyrosine kinases use similar patterns of activation of signaling pathways to mediate distinct biological outcomes of cell migration and proliferation, (2) NGF activates signaling proteins in smooth muscle cells similar to those activated during NGF-induced neuronal differentiation, and (3) the combinatorial effects of different signaling pathways are important for the regulation of smooth muscle cell migration and proliferation. Further studies using mutant trk receptors will help to define the signal transduction pathways mediating NGF-induced smooth muscle cell migration.

Pharmacokinetics and consistency of pericardial delivery directed to coronary arteries: direct comparison with endoluminal delivery.

BACKGROUND AND HYPOTHESIS: Pharmacologic modulation of the contents of the pericardial space has been shown to influence the response of coronary arteries to balloon injury. Endoluminal (EL) local delivery of various drugs into coronaries has been found to be limited by short residence time, as well as by highly variable deposited agent concentration. We hypothesized that compounds placed into the pericardial space (P) would penetrate into coronary tissue with greater consistency than seen after EL delivery and provide for prolonged coronary exposure to agents. METHODS AND RESULTS: 125I-labeled basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), albumin, or 131I-labeled diazeniumdiolated albumin (NONO-albumin) were delivered as model/therapeutic proteins into the porcine pericardial space (n = 15 pigs) or into coronaries using an EL delivery catheter (n = 48 arteries). In subjects receiving 125I-labeled proteins, the delivery target or mid-regions of the left anterior descending (LAD) and left circumflex (LCx) arteries were harvested at 1 h or 24 h for gamma-counting and autoradiography, and fractional intramural delivery (FID) or retention measured as percent agent in 100 mg artery/agent in infusate for both time points. In the animals receiving 131I-labeled NONO-albumin, serial gamma imaging was employed to evaluate the rate of redistribution in individual animals following either pericardial or endoluminal delivery. At 1 h, FID values ranged from 0.00064 to 0.0052% for P delivery (median 0.0022%), and from 0.00021 to 6.7 for EL delivery (median 0.27%). At 24 h, FID values ranged from 0.00011 to 0.003 for P delivery (median 0.0013), and from 0.0002 to 1.4 for EL delivery. The estimated T1/2 for bFGF redistribution from the vascular tissue was 22 h (P) and 7 h (EL), respectively, while the directly determined T1/2 values for NONO-albumin redistribution from the delivery region were 22.2 h (P) and 2.5 h (EL). CONCLUSIONS: These data show that pericardial fluid contents can access coronary arteries with intramural concentrations which typically vary by 10-15-fold, while EL delivery results in a remarkably wide intramural concentration range with up to 33,000-fold variability. The apparent redistribution rate is more rapid following EL delivery, possibly due to sustained diffusive tissue loading from the pericardial space. Pericardial delivery appears to offer substantial advantages over EL administration with respect to residence time and reproducibility.

Efficient in vivo catheter-based pericardial gene transfer mediated by adenoviral vectors.

Adenoviral vectors are promising agents for a number of in vivo gene therapy applications including diseases of the heart and coronary vessels. Efficient intravascular gene transfer to specific sites has been achieved in occluded vessels, but otherwise is hampered by the effect of blood flow on localized vector uptake in the vessel wall. An alternative delivery approach to coronary arteries is the expression of diffusible gene products into the pericardial space surrounding the heart and coronary arteries. However, in vivo pericardial access is comparatively difficult and has been limited to surgical approaches. We hypothesized that efficient adenovirus-mediated gene expression in pericardial lining mesothelium could be achieved by transmyocardial vector delivery to the pericardium. To evaluate this concept, a hollow, helical-tipped penetrating catheter was used to deliver vector-containing fluid directly into the intrapericardial space. The catheter was introduced percutaneously in anesthetized mongrel dogs, advanced into the right ventricle, and the tip passed through the apical right ventricular myocardium under direct radiographic visualization until the open end of the catheter tip resided in the intrapericardial space. Adenoviral vectors expressing either nuclear-localizing beta-galactosidase, cytoplasmic luciferase, or secreted human alpha 1AT reporters (Av1nBg, Av1Lu, or Av1Aa, respectively) were instilled through the catheter into the intrapericardial space. Three days later the animals were sacrificed and reporter gene expression was evaluated in pericardium, epicardium, and multiple other tissues. In animals receiving Av1nBg, beta-galactosidase activity was evident in most of the pericardial lining endothelium, up to 100% in many areas. In animals receiving Av1Lu, luciferase reporter activity was abundant in pericardial tissues, but near-background levels were observed in other organs. In animals receiving Av1Aa, human alpha 1AT was abundant (16-29 mg/ml) in pericardial fluid, but was undetectable in serum. All animals tolerated the procedure well with no electrocardiographic changes and no clinical sequelae. These observations demonstrate highly efficient adenovirus vector delivery and gene transfer and expression in the pericardium and support the feasibility of localized gene therapy via catheter-based pericardial approaches. We suggest that the pericardial sac may serve as a sustained-release protein delivery system for the generation of desired gene products or their metabolites for diffusion into the epicardial region.

Establishment of a clinically correlated human pericardial fluid bank: evaluation of intrapericardial diagnostic potential.

The development of a clinically correlated human pericardial fluid bank and database is described. A unique feature of this registry is the availability of a large number of pericardial fluid samples for testing with respect to multiple factors and for correlation with angiographic findings and clinical syndromes expressed by the patients. The collection of data at the present time comprises frozen pericardial fluid samples obtained from patients who have undergone cardiac surgery; and historical, clinical, and laboratory data obtained from the patient records. Nearly 400 samples have been stored and analyzed thus far, with sample entry continuing. This registry is designed to evaluate the local factors that play a role in mediating or reflecting myocardial or coronary responses. Pathophysiologic processes of particular interest include restenosis, plaque ruptures, and angiogenesis. Study of the pericardial fluid bank should lead to enhanced understanding of molecular mechanisms, as well as to the explanation for the reasons underlying interpatient variability in these processes. It is further anticipated that this information might provide a foundation for the diagnostic use of pericardial fluid to individualize therapies targeting angiogenesis or plaque physiology.

Gene therapy for restenosis: getting nearer the heart of the matter.

Intensive work over the past decade has been directed to the study of vascular gene transfer as an approach to the unresolved problem of restenosis. This effort has resulted in a significant foundation of knowledge relative to the activities of potentially therapeutic gene products as well as the capabilities and limitations of vector systems and mechanical delivery modalities available for effecting the vascular expression of these gene products. In several instances, significant progress has been made by experiments highlighting unexpected difficulties and the need for more comprehensive understanding. It is thus now possible to clearly define and address specific challenges that must be overcome in order to make feasible progress from the preclinical to the clinical arena. The key challenges at present appear to include the evolution of clinically practical delivery methods that meet the kinetic requirements of achieving efficient gene transduction and the availability of vectors that maximize efficiency while minimizing undesirable host responses. Emerging data suggest that approaches to solving each of these issues may have recently been developed. Basic research evaluating these new delivery mechanisms and molecular vectors is essential to establish their true potential for use in the clinical arena.

Activation of MAP kinase in vivo follows balloon overstretch injury of porcine coronary and carotid arteries.

Vascular restenosis involves contraction, proliferation, and remodeling of the arterial wall in response to overstretch injury. Mitogen-activated protein kinases (MAPKs) are implicated in both contraction and proliferation of vascular smooth muscle (VSM), and studies of porcine carotid arterial muscle strips have shown that mechanical stretch leads to the activation of the extracellular signal-regulated kinase (ERK) family of MAPKs in vivo. We, therefore, analyzed the acute effect of mechanical overstretch injury on ERK-MAPK (herein referred to simply as MAPK) activity in porcine coronary and carotid arteries in vivo. Balloon angioplasty catheters were inflated to 6 atm three times over 5 minutes at a balloon-artery ratio of 1.2:1 in either porcine coronary or carotid arteries. The arteries were snap-frozen after angioplasty, and MAPK activity was measured. Angioplasty of the left anterior descending (LAD, n = 5), left circumflex (LCx, n = 5), and carotid (n = 5) arteries effected an increase in MAPK activity compared with the activity in uninstrumented right coronary arteries (RCAs) or carotid arteries from the same animals used for controls. Balloon angioplasty of carotid arteries led to an increase in MAPK activity that was 7.7-fold over the activity in control arteries and comparable to the activity in stretched carotid arterial muscle strips in vivo. The increase in coronary artery kinase activity on angioplasty was variable from animal to animal. The increase in MAPK activity over that in control arteries ranged from 4.5- to 31.7-fold (mean +/- SEM, 10.7 +/- 5.3) in the LAD and 1.8- to 31.3-fold (mean +/- SEM, 9.7 +/- 5.7) in the LCx. There were no apparent inherent differences in the levels of MAPK activity in the three different types of coronary arteries (RCA, LAD, and LCx) without instrumentation. MAPK activation occurs rapidly during angioplasty, suggesting that this kinase may play an early role in initiating the injury response in both porcine coronary and carotid arteries. MAPKs may be key enzymes targeted to treat or prevent restenosis.