Nuclear accumulation of exogenous DNA fragments in viable cells mediated by FGF-2 and DNA release upon cellular injury.

We and others have previously shown that basic fibroblast growth factor (FGF-2 or bFGF) can be used as a targeting molecule to help carry plasmid DNA into cells when the growth factor molecule is physically coupled to the DNA molecule being delivered. Herein we report our observations on the FGF-mediated uptake of exogenous labeled DNA into cultured cells in a manner that is representative of that which may occur under physiological conditions at sites of wounded tissue. Cellular debris at such sites contains nucleic acid fragments released from dead cells, as well as growth factors such as FGF-2 that function early in the wound repair process. Using a cell culture model designed to mimic the local environment of a wound with respect to the presence of soluble FGF-2 and DNA fragments, we have shown that FGF-2 is able to direct the cellular uptake and nuclear localization of fragments of exogenous DNA via the FGF receptor into intact and healthy cells. Furthermore, we can monitor and quantitate this type of FGF-mediated DNA delivery by using indirect immunofluorescence of bromodeoxyuridine-labeled exogenous DNA. Our results suggest that this type of FGF-mediated DNA fragment uptake could allow for the transduction of viable nearest neighbor cells at sites of injury in vivo. Such a phenomenon may lead to mutational aberrations in the recipient cells and enhance the probability of wound carcinogenesis.

Proteinuria in a placebo-controlled study of basic fibroblast growth factor for intermittent claudication.

Intermittent claudication is the most common symptom of peripheral arterial disease (PAD), in part due to an inadequate rise in limb blood flow with exercise. Claudication causes a severe impairment in functional capacity and quality of life in over 3 million Americans. Basic fibroblast growth factor (bFGF) stimulates angiogenesis in vivo and improves limb blood flow in several animal models of hindlimb ischemia. However, the relative safety and efficacy of angiogenic molecules in the treatment of claudication has not been fully evaluated in prospective, blinded clinical trials. In this study, a randomized, double-blind, placebo-controlled, phase II trial of recombinant human bFGF for the treatment of intermittent claudication was performed. bFGF was administered weekly by intravenous infusions of 2 microg/kg for 6 sequential weeks (total dose 12 microg/kg). The primary efficacy endpoint was change in peak walking time (PWT) on a graded exercise treadmill protocol. Secondary efficacy endpoints included changes in functional status as measured by validated questionnaires. The study was stopped prematurely after treatment of the first 24 subjects due to proteinuria in five of the 16 subjects who received systemic bFGF, which exceeded 1000 mg/24 h in four of these five subjects. The small sample size limited evaluation of the predefined efficacy endpoints; however, there was no significant difference between the treatment and control groups for any of the measures of efficacy. In conclusion, intravenous administration of bFGF delivered at low doses weekly for 6 weeks was associated with a high rate of severe proteinuria. It is speculated that bFGF-related proteinuria in this study was primarily related to the systemic route of administration and the frequent dosing schedule. Future clinical trials of bFGF protein should carefully monitor renal function and consider alternative dosing schedules and drug administration routes.

Association of influenza vaccination and reduced risk of recurrent myocardial infarction.

BACKGROUND: Numerous studies have suggested that microbial agents may promote atherosclerosis. A smaller body of research has suggested that acute respiratory infection may be a risk factor for myocardial infarction (MI). We hypothesized that influenza vaccine might reduce the risk of recurrent MI in patients with documented coronary heart disease (CHD). METHODS AND RESULTS: A case-control study was performed on 218 CHD patients seen at Memorial Hermann Hospital during the influenza season of October 1997 through March 1998. Patients who experienced new MI were included in the case group, and those who did not experience new MI or unstable angina were assigned to the control group. Data were collected by structured review of patients' charts and through a subsequent telephone survey. Adjusted for history of influenza vaccination in previous years, multivariate logistic regression revealed risk of MI to be associated with current hypertension (OR 4.96, 95% CI 2.06 to 11.96, P<0.0001), hypercholesterolemia (OR 4.08, 95% CI 1.67 to 9.99, P=0.002), smoking (OR 3.75, 95% CI 1.76 to 7.98, P=0.001), and influenza vaccination (OR 0.33, 95% CI 0.13 to 0.82, P=0.017). Despite significant association in univariate analysis, multivitamin therapy and physical exercise were not associated with risk of reinfarction in multivariate analysis. CONCLUSIONS: In this study in patients with chronic CHD, vaccination against influenza was negatively associated with the development of new MI during the same influenza season. However, to address causal inference, examination of prospective data sets will be needed.

Thermal detection of cellular infiltrates in living atherosclerotic plaques: possible implications for plaque rupture and thrombosis.

BACKGROUND: Atherosclerotic lesions are heterogeneous and prognosis cannot easily be predicted, even with intracoronary ultrasound and angioscopy. Serial angiographic and necropsy studies suggest that the risk of plaque rupture correlates only weakly with the degree of stenosis. Most ruptured plaques are characterised by a large pool of cholesterol or necrotic debris and a thin fibrous cap with a dense infiltration of macrophages. The release of matrix-digesting enzymes by these cells is thought to contribute to plaque rupture. Other thromboses are found on non-ruptured but inflamed plaque surfaces. We postulated that both types of thrombotic events may be predicted by heat released by activated macrophages either on the plaque surface or under a thin cap. METHODS: To test the hypothesis, we measured the intimal surface temperatures at 20 sites in each of 50 samples of carotid artery taken at endarterectomy from 48 patients. The living samples were probed with a thermistor (24-gauge needle-tip; accuracy 0.1 degree C; time contrast 0.15 s). The tissues were then fixed and stained. FINDINGS: Plaques showed several regions in which the surface temperatures varied reproducibly by 0.2-0.3 degrees C, but 37% of plaques had substantially warmer regions (0.4-2.2 degrees C). Points with substantially different temperatures could not be distinguished from one another by the naked eye; such points could also be very close to one another (< 1 mm apart). Temperature correlated positively with cell density (r = 0.68, p = 0.0001) and inversely with the distance of the cell clusters from the luminal surface (r = -0.38, p = 0.0006). Most cells were macrophages. Infrared thermographic images also revealed heterogeneity in temperature among the plaques. INTERPRETATION: Living atherosclerotic plaques show thermal heterogeneity, which raises the possibility that an infrared catheter or other techniques that can localise heat or metabolic activity might be able to identify plaques at high risk of rupture or thrombosis.

Myocyte enhancer binding factor-2 expression and activity in vascular smooth muscle cells. Association with the activated phenotype.

Proliferation and phenotypic modulation of smooth muscle cells (SMCs) are major components of the vessel's response to injury in experimental models of restenosis. Some of the growth factors involved in restenosis have been identified, but to date little is known about the transcription factors that ultimately regulate this process. We examined the expression of the four members of the myocyte enhancer binding factor-2 (MEF2) family of transcription factors in cultured rat aortic SMCs (RASMCs) and a rat model of restenosis because of their known importance in regulating the differentiated phenotype of skeletal and cardiac muscle. In skeletal and cardiac muscle, the MEF2s are believed to be important for activating the expression of contractile protein and other muscle-specific genes. Therefore, we anticipated that the MEF2s would be expressed at high levels in medial SMCs that are producing contractile proteins and that they would be downregulated along with the contractile protein genes in neointimal SMCs. On the contrary, we observe that MEF2A, MEF2B, and MEF2D mRNAs are upregulated in the neointima, with the highest levels in the layer of cells nearest to the lumen, whereas MEF2C mRNA levels do not appreciably increase. Moreover, few cells in the media are making MEF2 proteins detectable by immunohistochemistry, whereas large numbers of neointimal cells are positive for all four MEF2s. These data suggest that the MEF2s are involved in the activated smooth muscle phenotype and not in the maintenance of contractile protein gene expression.

Stimulation of endothelial cells by doses of basic FGF-saporin that are lethal to smooth muscle cells.

Basic fibroblast growth factor (FGF) receptors are up-regulated in proliferating (vs. quiescent) aortic smooth muscle cells, according to the results of recent studies. This up-regulation allows the ribosome inactivator saporin (if linked to basic FGF) to enter and kill proliferating, but not quiescent smooth muscle cells in vitro and in vivo. The authors now report that endothelial cells exhibit a different response. In 10% serum, FGF-SAP (0.1-1 nM) stimulates protein synthesis and cell division in subconfluent endothelial cells, but inhibits protein synthesis and cell division in subconfluent smooth muscle cells. Endothelial cells were inhibited at 10 nM FGF-SAP. A stimulatory response was seen in smooth muscle cells only at 0.1 nM FGF-SAP, and only after serum deprivation. Both cell types were resistant to FGF-SAP at high cell density. These responses correlated with FGF receptor density, which was sixfold higher in smooth muscle than endothelial cells and twice as high in serum-free smooth muscle cells as in serum-deprived smooth muscle cells. Moreover, a dose of FGFSAP that inhibited neointimal smooth muscle accumulation after balloon injury did not inhibit reendothelialization. Thus, there is a dose range at which FGF-SAP has unique properties that may make it useful in the treatment of vascular injury.

Endogenous and exogenous nitric oxide protect against intracoronary thrombosis and reocclusion after thrombolysis.

BACKGROUND: Nitric oxide (NO), an endothelium-derived relaxing factor, plays an important role in regulating platelet activation. We evaluated the effect of NO in a canine model of intracoronary thrombosis, thrombolysis, and reocclusion. METHODS AND RESULTS: Before thrombosis was induced, 34 anesthetized dogs were treated with a continuous intracoronary infusion of saline (n = 8); NG-nitro-L-arginine (L-NNA, n = 8), an inhibitor of NO synthetase; L-arginine (n = 7), the precursor for NO; or sodium nitroprusside (SNP, n = 11), an NO donor. Ten minutes after the infusion was begun, an electric current of 150 microA was applied to the endothelium of coronary arteries to induce thrombosis. Occlusive thrombi developed in all dogs in the saline group (38 +/- 4 minutes) and the L-NNA group (30 +/- 6 minutes), in 6 of 7 dogs in the L-arginine group (81 +/- 18 minutes), and in 6 of 11 dogs in the SNP group (102 +/- 21 minutes) (P < .01). The time to thrombus was prolonged by L-arginine (P < .05) and SNP (P < .01). After 3 hours of thrombus formation in coronary arteries, tissue plasminogen activator and heparin were administered intravenously. Thrombi were lysed in 4 (of 8) dogs in the saline group (71 +/- 8 minutes), in 4 (of 8) dogs in the L-NNA group (72 +/- 8 minutes), in 4 (of 6) dogs in the L-arginine group (50 +/- 14 minutes), and in 4 (of 6) dogs in the SNP group (49 +/- 11 minutes) (P > .05). After thrombolysis, coronary artery reocclusion developed in all reperfused dogs in the saline group (30 +/- 8 minutes) and in the L-NNA group (48 +/- 12 minutes), in 3 (of 4) reperfused dogs in the L-arginine group (123 +/- 26 minutes), and in 3 (of 4) reperfused dogs in the SNP group (128 +/- 19 minutes) (P < .01). The ex vivo platelet aggregation induced by collagen was inhibited after in vivo treatment with L-arginine or SNP. CONCLUSIONS: Increasing NO production or giving an NO donor may inhibit platelet aggregation and delay intracoronary thrombus formation and reocclusion after thrombolysis.