Induction of angiogenesis by lidocaine and basic fibroblast growth factor: a model for in vivo retroviral-mediated gene therapy.

A strategy of direct, in vivo retroviral-mediated gene therapy targeting capillary endothelial cells must provide an environment of active angiogenesis. Both lidocaine and basic fibroblast growth factor (bFGF) promote angiogenesis, but the angiogenic response invoked by these substances in normal skeletal muscle has not been fully characterized. We sought to characterize these agents' angiogenic effects in anterior tibialis muscles of male Sprague-Dawley rats. An injection of either 1% lidocaine with 1:100,000 epinephrine or alternate-day injections of bFGF (0.025 or 0.25 microgram) with or without heparin were tested (n = 6 muscles/condition). Rats were sacrificed 4, 7, 10, or 12 days later and muscles were evaluated histologically to determine the number of proliferating cells using 5-bromo-2'-deoxycytidine (BrdC) and evaluated for capillary density using Griffonia simplicifolia I (GSI) lectin. At all time points, lidocaine produced at least 20-fold greater capillary density and cellular proliferation than PBS control (P < 0.0001). Injections of high-dosage bFGF produced more than fivefold greater capillary density than control injections at 7 and 10 days (P < 0.001), and more than twofold greater proliferation at 4, 7, and 12 days (P < 0.001). Capillary density returned to control levels 12 days following bFGF administration, whereas it remained well above control levels for 12 days after lidocaine administration. To confirm that lidocaine can be utilized in gene therapy strategies targeting vascular endothelium and skeletal muscle fibers, concentrated pLJ retrovirus containing cDNA for the heat-stable human placental alkaline phosphatase (hpAP) marker gene was infused into the rat hindlimb vasculature 4 days post-lidocaine administration. Rats receiving pLJhpAP retrovirus demonstrated significant hpAP transgene expression in endothelial cells and myocytes 21 days after the lidocaine injection (n = 6 muscles). In contrast, controls receiving pLJhpAP infusion without prior lidocaine administration failed to demonstrate any hpAP transgene expression. Lidocaine treatment evokes a substantially higher proliferative response than bFGF and, importantly, a durable angiogenic response in skeletal muscle. Thus, lidocaine is an ideal agent to induce angiogenesis in preparation for direct in vivo retroviral-mediated gene therapy targeting vascular endothelium.

Transplantation of lac-Z-transduced microvascular endothelial cells into the skeletal muscle capillary bed of the rat hindlimb occurs independent of the duration of femoral artery occlusion after injection of cells.

The skeletal muscle capillary bed may be an ideal recipient site for transplantation of genetically modified autologous endothelial cells and thus provide a basis for a technique of somatic gene therapy that would be applicable to a variety of acquired and inherited human diseases. The purpose of this study was to test the hypothesis that adhesion of lac-Z-transduced microvascular endothelial cells (MVEC) in the skeletal muscle capillary bed in vivo is dependent on the duration of arterial occlusion after injection of the transduced MVEC. MVEC derived from the abdominal fat pad of syngeneic rats (Wistar F-455) were transfected with the BAG vector, a replication-incompetent retroviral vector containing the lac-Z gene for beta-galactosidase and the Tn5 gene for selection of the transduced cells by the neomycin analogue, G418. lac-Z-transduced MVEC were radiolabeled with 125I-PKH-95, and, after the femoral artery was occluded for 10 min, these cells (1 to 2 x 10(6)) were injected intraarterially into the rat hindlimb. In the experimental groups the femoral artery clamp was removed at 0, 60, or 120 min after injection. A control group without pre- or postinjection femoral arterial occlusion was also studied. Adhesion of MVEC in the skeletal muscle capillary bed (mean percentage of injected 125I activity) was determined in groups of 4 rats at 1 day, 1 week, and 1 month after injection. Adhesion of the transduced MVEC did not increase as the duration of femoral artery occlusion after injection was increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Adhesion and incorporation of lacZ-transduced endothelial cells into the intact capillary wall in the rat.

Use of the capillary bed of skeletal muscle as an in vivo recipient site to transplant autologous endothelial cells that have undergone gene transfer ex vivo has considerable potential as a technique of somatic gene therapy. Here we document a previously unrecognized capacity of endothelial cells to adhere and incorporate spontaneously into confluent endothelial cell monolayers in vitro and in vivo. This spontaneous adhesion and incorporation of endothelial cells enabled us to seed lacZ-transduced endothelial cells into the wall of skeletal muscle capillaries of the hindlimb of the rat. Certain transduced endothelial cells became incorporated within the capillary wall, whereas others remained within the capillary lumen where they formed focal, electron-dense, contacts with host endothelium. lacZ expression in the capillary bed was documented for up to 1 month after transplantation. Use of the intact capillary bed of skeletal muscle as an in vivo recipient site for transduced, autologous endothelial cells holds promise as a strategy for somatic gene therapy to treat various genetic and acquired human diseases.

ATLAS-plus: multimedia instruction in embryology, gross anatomy, and histology.

ATLAS-plus [Advanced Tools for Learning Anatomical Structure] is a multimedia program used to assist in the teaching of anatomy at the University of Michigan Medical School. ATLAS-plus contains three courses: Histology, Embryology, and Gross Anatomy. In addition to the three courses, a glossary containing terms from the three courses is available. All three courses and the glossary are accessible in the ATLAS-plus environment. The ATLAS-plus environment provides a consistent set of tools and options so that the user can navigate easily and intelligently in and between the various courses and modules in the ATLAS-plus world. The program is a collaboration between anatomy and cell biology faculty, medical students, graphic artists, systems analysts, and instructional designers.

The role of an endothelial cell lining in limiting distal anastomotic intimal hyperplasia of 4-mm-I.D. Dacron grafts in a canine model.

The effect of an endothelial cell (EC) lining on intimal hyperplasia at the distal anastomosis of Dacron grafts was assessed in a canine model. Enzymatically derived autologous EC were used to seed 14 to 17 cm long, 4 mm I.D., knitted Dacron aortoiliac grafts implanted in an end-to-side manner in six dogs (Group I). Unseeded grafts were similarly implanted in six control dogs (Group II). All animals received acetylsalicylic acid (325 mg po qd) 24 h prior to graft placement and for 2 weeks postoperatively. Distal anastomotic intimal hyperplasia (AIH) and luminal surface EC coverage were quantitated at the conclusion of a 16-week study period. Patency for Group I and Group II grafts were 90% and 55%, respectively (p = 0.07). Maximum AIH, defined as the maximum reduction of luminal cross-sectional area at the distal anastomosis, was not significantly different between Group I (13.1 +/- 8.0%) and Group II (15.1 +/- 7.3%) conduits. However, AIH was inversely related to the extent of luminal EC coverage (r = -0.6, p less than 0.05), thus greater endothelialization was associated with decreased AIH. These data support the idea that EC coverage of the luminal surface of prosthetic vascular grafts may limit the development of AIH.

Extracellular matrix preparation of expanded polytetrafluoroethylene grafts seeded with endothelial cells: influence on early platelet deposition, cellular growth, and luminal prostacyclin release.

The importance of blood and extracellular matrix precoating of expanded polytetrafluoroethylene (ePTFE) grafts on the effectiveness of endothelial cell (EC) seeding was assessed in a canine experimental model. Part I of the study documented ex vivo platelet deposition in 256 ePTFE grafts, 6 cm x 4 mm internal diameter, after implantation as femoral artery-femoral vein or carotid artery-jugular vein arteriovenous shunts. These conduits were precoated with blood, fibronectin, laminin, or collagen type IV with laminin, after which they were seeded with enzymatically derived and cultivated venous canine endothelium at a density of 30,000 to 40,000 EC/cm2 of graft surface. Luminal deposition of Indium 111-labeled platelets, expressed as 10(8) platelets/cm2, at 30 minutes (n = 176) and 24 hours (n = 80), respectively, was 2.29 and 0.30 for blood, 2.83 and 0.37 for fibronectin, 0.99 and 0.08 for laminin, and 0.98 and 0.11 for collagen type IV with laminin. Part II of the study documented in vivo luminal EC coverage at 14 days of 6 cm x 4 mm internal diameter ePTFE femoral or carotid arterial grafts (n = 8) prepared in the same manner as part I ex vivo shunt grafts. EC coverage with blood, fibronectin, laminin, and collagen type IV with laminin preparation was 42%, 49%, 44%, and 52%, respectively. The graft:carotid artery ratio of luminal 6-keto-PGF1 alpha release at 14 days with these same four preparations was 0.38, 0.31, 0.35, and 0.32, respectively. Precoatings of ePTFE prostheses with fibronectin, laminin, and collagen type IV are known to enhance the initial attachment of seeded EC. Fibronectin caused an insignificant increase in early platelet accumulation; laminin or laminin with collagen type IV preparations were associated with significantly less (p less than 0.005) deposition of platelets when compared to whole blood preparations. Most importantly, none of the four preparation techniques resulted in different in vivo rates of EC growth or luminal release of prostacyclin from conduits studied 14 days after implantation.

Effects of plasma on PGI2 release from prolonged perfused canine veins.

Prostacyclin release from endothelial cells in culture appears increased by the presence of plasma, but occurrence of a similar phenomenon in intact vessels has not been established. In the present investigation release of 6-keto-PGF1 alpha, the stable breakdown product of prostacyclin, was quantitated from canine veins perfused ex vivo for 15 minute periods, using three different perfusates: 1) Hank's balanced salt solution (HBSS), 2) 20% platelet poor plasma (PPP) derived from heparinized blood, in HBSS, and 3) 20% PPP in HBSS with added arachidonic acid (AA). No differences in initial 6-keto-PGF1 alpha release existed among the three perfusates. However, PPP and PPP + AA solutions were associated with lesser declines in release, than occurred with HBSS alone, these differences being statistically significant after 60 min of perfusion (p less than 0.05). When PPP derived from heparinized and citrated blood rather than from only heparinized blood was used, there was a significantly lower release of prostacyclin (p less than 0.05). The latter may be due to citrate binding of calcium. These data indicate that autologous plasma does not alter initial prostacyclin release from freshly harvested canine veins, but that it weakens the decline in release usually following prolonged periods of perfusion.

Small-caliber polyurethane and polytetrafluoroethylene grafts: a comparative study in a canine aortoiliac model.

In vivo stability of a new small-caliber polyurethane graft (n8) was assessed in a canine aortoiliac model and compared to that of a conventional expanded polytetrafluoroethylene (ePTFE) graft (n8). Six months following implantation, marked aneurysmal dilatation to 230 +/- 80% (mean +/- SD) of the original diameter occurred in polyurethane grafts, while dilatation to 110 +/- 8% of the original diameter occurred in ePTFE grafts (p less than 0.005). Interval patency was 75% for each graft type. Luminal thrombus affected 59% of polyurethane graft surfaces compared to 22% of ePTFE graft surfaces (p less than 0.01). Qualitative examination of representative sections of polyurethane conduits demonstrated thick inner capsules with numerous small islands of graft material surrounded by macrophages and bands of mature fibrous tissue, in contrast to the thinner neointima and limited anastomotic pannus ingrowth observed in ePTFE grafts.

Prostanoid release from ex vivo perfused canine arteries and veins: effects of prolonged perfusion, intermittent perfusion, as well as exposure to exogenous arachidonic acid, thrombin and bradykinin.

Regulation of prostanoid release from ex vivo perfused vessel segments is not fully understood. A series of perfusion experiments were performed with canine arteries and veins to define certain regulatory phenomena. Arteries were perfused with pulsatile flow of 90 ml/min at a pressure of 100 mmHg, and veins with nonpulsatile flow of 90 ml/min at a pressure of 7 mmHg. Segments were perfused with Hanks' balanced salt solution for five 15-min periods with the perfusate exchanged after each study period. With onset of perfusion, there was an initial burst of prostacyclin release to 127 +/- 40 pg/mm2, declining to 32 +/- 10 pg/mm2 after 60 minutes (p less than 0.005). If perfusion continued for 5.5 hours, there was a stable release period between 1 and 3 hours, followed by a very slow decline. At that time addition of arachidonic acid (AA) increased prostacyclin release six-fold (p less than 0.01). Vessels perfused for 1 hour and then rested for another hour, responded to reperfusion at the second onset of flow with a two-fold increase in prostacyclin release (p less than 0.01). Vessels perfused with thrombin, bradykinin or AA (either added to each perfusate or only to the last perfusate) exhibited greater prostacyclin release than did control segments. Release of thromboxane steadily declined with time in all parts of the study, and only increased with the addition of AA to the perfusate.(ABSTRACT TRUNCATED AT 250 WORDS)

Protamine-induced reductions of endothelial cell ATP.

Protamine, a polycationic protein used to reverse heparin anticoagulation, is frequently associated with decreased oxygen consumption, systemic hypotension, pulmonary artery hypertension, and bradycardia. This investigation examines the hypothesis that these events reflect toxic effects of protamine on endothelial cells. Cultured bovine pulmonary artery endothelium was exposed to protamine (12.5 to 500 micrograms/ml, corresponding to clinical doses 0.75 to 30 mg/kg), either alone (n = 6) or 3 minutes after exposure to heparin, 0.1 IU/microgram protamine (n = 6). ATP was measured 1 to 180 minutes after protamine by a luciferase-luciferin assay and cell viability determined by trypan blue exclusion. Ultrastructure was assessed by transmission electron microscopy. Polylysine, 25 micrograms/ml, a cytotoxic polycationic agent, was also studied. Dose-dependent reductions in ATP (range, -11% to -51%) and ATP per viable cell (up to -41%) occurred. Decreases in ATP did not occur until after 30 minutes with protamine alone, compared with differences as early as 1 minute after protamine with prior heparin. Progressive mitochondrial injury was noted evident by swollen cristae, vacuolization, and eventual disruption. Polylysine caused similar changes. Protamine decreases endothelial cell ATP and prior heparin exposure accelerates this effect. The toxicity may reside in the positive charges on these molecules and mitochondrial damage may account for reductions in cellular ATP and systemic oxygen consumption.

Effects of duration of acetylsalicylic acid administration on patency and anastomotic hyperplasia of ePTFE grafts.

Effects of duration of acetylsalicylic acid (ASA) administration on the patency and development of distal anastomotic intimal hyperplasia (DAIH) of endothelial cell (EC) seeded and unseeded prosthetic aortoiliac grafts were studied in a canine model. ASA, 5 gr po qd, was administered to dogs 1 day prior to placement of bilateral, 12 to 17 cm long, 5 mm inside diameter expanded polytetrafluoroethylene (ePTFE) aortoiliac grafts and continued for 2 wk (Group 1, n = 12 dogs) or 16 wk (Group 2, n = 12 dogs). Six dogs in each group received autologous EC seeded grafts, while the others received unseeded grafts. Prosthesis patency was assessed weekly. At the conclusion of the study, DAIH was measured on serial sections using a computer-linked digitizer. The 16 wk patency for Group 1 grafts was 67%, while that for Group 2 grafts was 88% (p less than 0.09). Luminal narrowing due to DAIH was not significantly different between Groups 1 and 2 (7.7 +/- 8.3% [means +/- SD] and 9.0 +/- 7.8% respectively). EC seeding improved the 16 wk combined patency from 62 to 92% (p less than 0.02). A more complete luminal endothelial cell lining was correlated with reduced DAIH (r = -0.4, p less than 0.05). Chronic ASA administration prevented graft thrombosis between 2 and 4 wk postimplantation in this study but was not associated with decreased DAIH.

Arterial 6-keto-PGF1 alpha and TxB2 release in ex vivo perfused canine vessels: effects of pulserate, pulsatility, altered pressure and flow rate.

Certain experimental conditions are known to influence the release of prostacyclin and thromboxane from the vessel wall. The specific effects of altered pulsatility, pressure, and flow rate on intraluminal release of 6-keto-PGF1 alpha and thromboxane B2 were assessed in canine arteries perfused ex vivo for five 15 min periods with arachidonic acid (AA) added during the last period. Control arteries were perfused at 100 mmHg with pulsatile flow of 90 ml/min. Experimental arteries were perfused at 7, 50 and 200 mmHg with pulsatile flow of 90 ml/min, and at 100 mmHg pressure with pulsatile flow of 20, 60, 130 and 180 ml/min, as well as at 100 mmHg with 90 ml/min nonpulsatile flow. Perfusion pump rates of 44 and 96 beats/min were also assessed. The lowest perfusion pressure, 7 mmHg, resulted in a lesser initial release of prostacyclin compared to higher pressures, and there was a tendency to a higher release of prostacyclin with increasing pressures. There was also a tendency for a lesser response to AA in arteries perfused at 200 mmHg, perhaps due to endothelial cell damage. Nonpulsatile flow was associated with a decreased initial release of prostacyclin, and diminished release following addition of AA when compared to pulsatile flow. Altered flow rate elicited no difference in prostacyclin release, although there was a tendency towards a lesser release when perfused at 20 ml/min compared to 130 ml/min or 180 ml/min. Thromboxane release was decreased by nonpulsatile flow but was otherwise unaffected by the experimental conditions tested. It is concluded that pulsatility enhances release of prostacyclin from arteries.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of thromboxane synthetase inhibition on patency and anastomotic hyperplasia of vascular grafts.

The efficacy of a thromboxane synthetase inhibitor (U-63,557A, Upjohn) in promoting early patency and inhibiting anastomotic intimal hyperplasia in ePTFE grafts was compared to that of acetylsalicylic acid (ASA) in a canine model. Animals were started on ASA 5 gr po qd (Group I, n = 12) or U-63,557A 10 mg/kg po bid (Group II, n = 12) 1 day before placement of bilateral 5-mm-i.d., 13- to 16.5-cm-long ePTFE aortoiliac grafts and continued on the medication for the 16-week study. Six dogs in each group received autologous endothelial cell-seeded grafts, while the other six received unseeded grafts. Patency was determined weekly by assessment of femoral pulses. At the conclusion of the study anastomotic intimal hyperplasia was measured on serial sections through the distal anastomosis using a computer-linked digitizer. In Group I the patencies of seeded and unseeded grafts were not significantly different, being 100 and 83%, respectively. Furthermore, luminal narrowing due to intimal hyperplasia was not significantly different being 9.1 +/- 7.6% (chi +/- SD) in seeded grafts and 8.8 +/- 8.1% in unseeded grafts. On the other hand, in Group II the seeded grafts had significantly improved patency when compared to the unseeded grafts (83% vs 33%, P less than 0.05) and less luminal narrowing (11.4 +/- 11.1% vs 21.9 +/- 19.5%, P less than 0.01). Although U-63,557A administration promoted patency of unseeded grafts compared to no antiplatelet medication (0% patency), it was significantly less effective than ASA in improving patency (P less than 0.05) and inhibiting luminal narrowing (P less than 0.01).

Karyotype analysis of cell sex to determine the source of vascular graft luminal linings following autologous and nonautologous endothelial cell seeding.

Endothelial cell-seeded bilateral carotid ePTFE grafts were studied in 11 dogs to determine the source of their eventual luminal linings. One graft was prepared with autologous cells and the other with nonautologous cells. Immunosuppression was accomplished with cyclosporin and prednisolone. At 2 and 3 weeks post implantation, grafts were removed, revealing 9 of 11 autologous and 8 of 11 nonautologous seeded conduits to be patent. Linings were harvested enzymatically and grown in tissue culture, with karyotype analysis limited to cultures that exhibited greater than or equal to 50% endothelium (n5 autologous, n7 nonautologous). Sex type was classified in 20 cells from each graft. All five autologous cell-seeded grafts revealed host cells. Among the seven nonautologous cell-seeded grafts, one harbored only non-autologous cells, three revealed only autologous cells, and three exhibited a predominance of autologous cells with lesser numbers of nonautologous endothelium. All 100 cells from the autologous-seeded grafts were the same sex as seeded cells, while only 28 of the 140 cells from the nonautologous seeded grafts were the same sex as seeded cells (P less than 0.001). Thus, nonautologous endothelial cell seeding with immunosuppression did not uniformly produce nonautologous cell linings, but was more often associated with autologous endothelial cell linings that may have been promoted by the nonautologous cell seeding.

Comparison of static incubation versus physiologic perfusion techniques for quantitation of luminal release of prostacyclin and thromboxane in canine arteries and veins.

Intraluminal release of 6-keto-PGF1 alpha and TxB2 in ex vivo canine arteries and veins was assessed during five consecutive 15-min periods using static incubation and physiologic perfusion techniques. Arterial segments were perfused with 90 ml/min pulsatile flow at 100 mm Hg and vein segments with 90 ml/min nonpulsatile flow at 7 mm Hg. During the final 15-min period vessels were stimulated with arachidonic acid (AAS). Perfusion of vein segments caused a higher release of 6-keto-PGF1 alpha during the first 30 min (P less than 0.05) and following AAS (P less than 0.05) than did static incubation. Perfused arterial segments exhibited a higher release than did incubated segments of 6-keto-PGF1 alpha for 45 min (P less than 0.01) as well as following AAS (P less than 0.01). TxB2 release was higher during the entire observation period in perfused arteries and veins compared to incubated vessels (P less than 0.01 and less than 0.05, respectively). There was no correlation between the amounts of 6-keto-PGF1 alpha or TxB2 released when comparing values obtained by one technique to values obtained by the other (P greater than 0.1). These data suggest that flow related shear stress alters vascular prostanoid production, and that such should be accounted for when interpreting results of studies on prostacyclin and thromboxane release from intact vessels.

Endothelial cell seeding of expanded polytetrafluoroethylene vena cava conduits: effects on luminal production of prostacyclin, platelet adherence, and fibrinogen accumulation.

The blood-surface interface of 12 mm ID x 5 cm ePTFE vena cava conduits, unseeded (n = 8) and seeded (n = 8) with enzymatically derived autologous endothelial cells, was studied in a canine model at 4 and 12 weeks after graft implantation. Acetylsalicylic acid (325 mg each day) and Coumadin (prothrombin time 1.4 to 1.7 times the control value) were administered preoperatively and continued 4 weeks postoperatively. Platelets labeled with 111In and 125I-labeled fibrinogen were administered 24 hours before graft removal. Luminal platelet adherence, expressed as 10(6) platelets/cm2 of graft surface, was 8.9 +/- 5.6 vs. 56.4 +/- 8.0 (p less than 0.008) and 4.0 +/- 0.9 vs. 12.4 +/- 2.3 (p less than 0.005) in seeded vs. unseeded grafts at 4 and 12 weeks, respectively. Luminal fibrinogen deposition, expressed in micrograms per square centimeter of graft surface, was 11.8 +/- 2.2 vs. 32.0 +/- 2.0 (p less than 0.06) and 6.1 +/- 2.4 vs. 12.4 +/- 6.3 (p less than 0.005) in seeded vs. unseeded grafts at 4 and 12 weeks, respectively. Cumulative 4- and 12-week luminal production of 6-keto-PGF1 alpha from seeded and unseeded grafts represented 11% and 5%, respectively, of that produced from the native iliac vein. Luminal endothelial cell coverage was 71% +/- 22% vs. 33% +/- 9% and 79% +/- 8% vs. 55% +/- 8% (p less than 0.05) in seeded and unseeded grafts at 4 and 12 weeks, respectively. Although endothelialization was not complete in seeded vena cava grafts, it is clear that seeded prostheses exhibited improved thromboresistance compared with unseeded conduits.

Lack of effect of dihydroergotamine on endothelial and smooth muscle cell proliferation and endothelial cell prostanoid production.

The most important effect of dihydroergotamine is venoconstriction, but certain metabolic effects and changes in vessel prostanoid activity have also been suggested. In this study endothelial cell production of 6-keto PGF1 alpha and TxB2 was quantitated in vitro. No evidence of altered prostanoid production was noted after incubation with dihydroergotamine (exposure ranging from 5 x 10(-3) to 5 x 10(-7) g/l). Similarly, no effect of dihydroergotamine on the growth rates of endothelial cells or smooth muscle cells in vitro was documented.