New methods for time-resolved fluorescence spectroscopy data analysis based on the Laguerre expansion technique--applications in tissue diagnosis.

OBJECTIVES: A new deconvolution method for the analysis of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) data is introduced and applied for tissue diagnosis. METHOD: The intrinsic TR-LIFS decays are expanded on a Laguerre basis, and the computed Laguerre expansion coefficients (LEC) are used to characterize the sample fluorescence emission. The method was applied for the diagnosis of atherosclerotic vulnerable plaques. RESULTS: At a first stage, using a rabbit atherosclerotic model, 73 TR-LIFS in-vivo measurements from the normal and atherosclerotic aorta segments of eight rabbits were taken. The Laguerre deconvolution technique was able to accurately deconvolve the TR-LIFS measurements. More interesting, the LEC reflected the changes in the arterial biochemical composition and provided discrimination of lesions rich in macrophages/foam-cells with high sensitivity (> 85%) and specificity (> 95%). At a second stage, 348 TR-LIFS measurements were obtained from the explanted carotid arteries of 30 patients. Lesions with significant inflammatory cells (macrophages/foam-cells and lymphocytes) were detected with high sensitivity (> 80%) and specificity (> 90%), using LEC-based classifiers. CONCLUSION: This study has demonstrated the potential of using TR-LIFS information by means of LEC for in vivo tissue diagnosis, and specifically for detecting inflammation in atherosclerotic lesions, a key marker of plaque vulnerability.

Diagnosis of vulnerable atherosclerotic plaques by time-resolved fluorescence spectroscopy and ultrasound imaging.

In this study, time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) and ultrasonography were applied to detect vulnerable (high-risk) atherosclerotic plaque. A total of 813 TR-LIFS measurements were taken from carotid plaques of 65 patients, and subsequently analyzed using the Laguerre deconvolution technique. The investigated spots were classified by histopathology as thin, fibrotic, calcified, low-inflamed, inflamed and necrotic lesions. Spectral and time-resolved parameters (normalized intensity values and Laguerre expansion coefficients) were extracted from the TR-LIFS data. Feature selection for classification was performed by either analysis of variance (ANOVA) or principal component analysis (PCA). A stepwise linear discriminant analysis algorithm was developed for detecting inflamed and necrotic lesion, representing the most vulnerable plaques. These vulnerable plaques were detected with high sensitivity (>80%) and specificity (>90%). Ultrasound (US) imaging was obtained in 4 carotid plaques in addition to TR-LIFS examination. Preliminary results indicate that US provides important structural information of the plaques that could be combined with the compositional information obtained by TR-LIFS, to obtain a more accurate diagnosis of vulnerable atherosclerotic plaque.

Application of the laguerre deconvolution method for time-resolved fluorescence spectroscopy to the characterization of atherosclerotic plaques.

This study investigates the ability of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) to detect inflammation in atherosclerotic lesion, a key feature of plaque vulnerability. A total of 348 TR-LIFS measurements were taken from carotid plaques of 30 patients, and subsequently analyzed using the Laguerre deconvolution technique. The investigated spots were classified as Early, Fibrotic/Calcified or Inflamed lesions. A stepwise linear discriminant analysis algorithm was developed using spectral and TR features (normalized intensity values and Laguerre expansion coefficients at discrete emission wavelengths, respectively). Features from only three emission wavelengths (390, 450 and 500 nm) were used in the classifier. The Inflamed lesions were discriminated with sensitivity > 80% and specificity > 90 %, when the Laguerre expansion coefficients were included in the feature space. These results indicate that TR-LIFS information derived from the Laguerre expansion coefficients at few selected emission wavelengths can discriminate inflammation in atherosclerotic plaques. We believe that TR-LIFS derived Laguerre expansion coefficients can provide a valuable additional dimension for the detection of vulnerable plaques.

Novel methods of time-resolved fluorescence data analysis for in-vivo tissue characterization: application to atherosclerosis.

This study investigates the ability of new analytical methods of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) data to characterize tissue in-vivo, such as the composition of atherosclerotic vulnerable plaques. A total of 73 TR-LIFS measurements were taken in-vivo from the aorta of 8 rabbits, and subsequently analyzed using the Laguerre deconvolution technique. The investigated spots were classified as normal aorta, thin or thick lesions, and lesions rich in either collagen or macrophages/foam-cells. Different linear and nonlinear classification algorithms (linear discriminant analysis, stepwise linear discriminant analysis, principal component analysis, and feedforward neural networks) were developed using spectral and TR features (ratios of intensity values and Laguerre expansion coefficients, respectively). Normal intima and thin lesions were discriminated from thick lesions (sensitivity >90%, specificity 100%) using only spectral features. However, both spectral and time-resolved features were necessary to discriminate thick lesions rich in collagen from thick lesions rich in foam cells (sensitivity >85%, specificity >93%), and thin lesions rich in foam cells from normal aorta and thin lesions rich in collagen (sensitivity >85%, specificity >94%). Based on these findings, we believe that TR-LIFS information derived from the Laguerre expansion coefficients can provide a valuable additional dimension for in-vivo tissue characterization.

Acute arterial thrombosis causes endothelial dysfunction: a new paradigm for thrombolytic therapy.

PURPOSE: The goals of this study were to delineate the time course of endothelial dysfunction after arterial thrombosis, to determine the cause of endothelial dysfunction in this setting, and to determine whether modulating standard thrombolytic therapy would ameliorate the thrombosis-mediated endothelial dysfunction. METHODS: Male adult rats underwent infrarenal aortic occlusion by means of clip ligature to induce arterial thrombosis. After 30 minutes, 1, 2, and 3 hours, ring segments from the infrarenal aorta were harvested and placed into physiologic buffer baths. With the use of a force transducer, both endothelial-dependent relaxation (EDR) and endothelial-independent relaxation (EIR) were measured. Endothelial function and presence were determined by means of factor VIII immunohistochemical staining. Endothelial morphology was evaluated with scanning electron microscopy (SEM). Nitric oxide (NO) levels were determined with a chemiluminescent assay of its nitrite/nitrate metabolites (NO(x)). Standard thrombolytic therapy with urokinase (UK) was infused into thrombosed aortic ring segments and compared with UK supplemented with both low-dose L -arginine (2 mmol) and high-dose L -arginine (20 mmol). RESULTS: Arterial thrombosis decreases EDR. The nadir of EDR occurs 1 hour after thrombosis (mean +/- SE, 13% +/- 6.4% vs 94% +/- 2.6% for controls, P <.005), with persistent lowering of EDR as long as 3 hours after thrombosis. EIR is preserved, and vasoconstriction with norepinephrine or potassium buffer is unaltered. Both endothelial function and presence (n = 6 per group) were documented by means of factor VIII immunohistochemistry. An intact monolayer of endothelium at all time intervals after thrombosis was revealed by means of SEM analysis. No differences between control and thrombosed specimens were revealed by means of the grading of SEM images. Local NO(x) levels were lower after 1 hour of thrombosis, with an increase higher than baseline values at 3 hours. The addition of low-dose L -arginine resulted in a minor increase in EDR. However, high-dose L -arginine resulted in a significant increase in EDR versus controls receiving UK alone (64% +/- 6.3% vs 38% +/- 4.4%, P <.05). Correspondingly, local NO(x) levels were 20-fold higher after the high-dose L -arginine supplementation when compared with UK thrombolysis alone (2.8 +/- 0.52 micromol/L vs 0.133 +/- 0.02 micromol/L, n = 6 samples/group, P <.005). CONCLUSION: Acute arterial thrombosis causes endothelial dysfunction, without causing endothelial cell loss. Endothelial function reaches a nadir after 1 hour of thrombosis. EIR and vasoconstriction remain unaffected, indicating normal smooth muscle cell function. NO(x) levels suggest that NO levels are decreased acutely after thrombosis. Supplementing standard thrombolytic therapy with the NO precursor, l-arginine, ameliorates the endothelial dysfunction seen after acute thrombosis by increasing local NO production.

Combined carotid endarterectomy and coronary bypass: a decade experience at UCLA.

PURPOSE: The purpose of this review was to determine outcomes for combined carotid endarterectomy (CEA) and coronary revascularization (CABG) in patients with asymptomatic carotid stenosis. METHODS: We reviewed the medical records of consecutive combined procedures (CEA and CABG), performed at UCLA Medical Center from October, 1989 to January, 1999. FINDINGS: There were 43 patients, 27 men and 16 women, with a mean age of 71 yr (range 51-87). Thirty-four patients 79% (34/43) had asymptomatic carotid stenosis. Stroke occurred in three patients (3/43 = 6.9%). Stroke ipsilateral to the CEA occurred in two patients: one asymptomatic (1/34 = 2.9%) and one symptomatic (1/9 = 11.1%). CONCLUSIONS: The majority of patients undergoing combined CEA/CABG have asymptomatic carotid stenosis identified in preparation for elective CABG. The asymptomatic carotid subset stroke rate of 2.9% resulting from a combined CEA/CABG is higher than our reported rate for CEA performed alone. In patients with asymptomatic carotid stenosis, the combined procedure should be selectively performed.

Dexamethasone inhibits the phosphorylation of retinoblastoma protein in the suppression of human vascular smooth muscle cell proliferation.

We have previously demonstrated that dexamethasone (DEX) suppresses neointimal hyperplasia and proliferation of rat aortic smooth muscle cells (SMC) by inducing a late G1 phase cell cycle arrest. Phosphorylation of retinoblastoma protein (Rb) regulates cell proliferation by controlling progression from G1 to S phase of the cell cycle. We hypothesized that DEX inhibits human vascular SMC proliferation and causes cell cycle arrest through inhibition of Rb phosphorylation. Human aortic SMC were cultured and treated with incremental doses of DEX. Cell counts and [(3)H]thymidine uptake were determined after 72 h. To examine the effects of DEX on the cell cycle, cells were synchronized by serum deprivation, restimulated to enter G1 phase, and treated with 10(-5) M DEX, and protein was extracted at sequential time points. Flow cytometry was performed to track cell cycle progression. Western blots were performed to examine Rb phosphorylation. DEX inhibited smooth muscle cell proliferation and DNA synthesis in a concentration-dependent fashion. Flow cytometry indicated that DEX induces a G1 phase cell cycle arrest. DEX inhibited the phosphorylation of Rb protein compared to control. DEX inhibits the proliferation of human vascular SMC by inducing G1 phase cell cycle arrest. DEX inhibited the phosphorylation of Rb, a key step in the progression of the cell from G1 to S phase. Elucidation of the mechanism of DEX may be helpful in treatment strategies for preventing neointimal hyperplasia as well as other disorders of cell proliferation.

Combined revascularization and microvascular free tissue transfer for limb salvage: a six-year experience.

Atherosclerotic vascular disease causing extensive tissue loss of the lower extremities often results in primary amputation. Combined revascularization and free tissue transfer has been described as a method of extending limb salvage to these patients. The durability of this combined procedure remains unknown, thus the objective of this report is to describe the immediate and long-term results in a series collected over 6 years. From 1992 to 1998, 15 patients with a mean age of 60 years underwent combined revascularization and free tissue transfer. Mean ulcer size measured 45 cm(2) for a mean duration of 7.4 months preoperatively and 12 patients had exposed bone or tendon. Vascular reconstruction included popliteal (3), tibial (6), and pedal (6) bypass with concomitant myocutaneous free flap, using mostly rectus abdominis or latissimus dorsi muscle. There were no perioperative deaths. One patient suffered a nonfatal myocardial infarction. Two patients had a postoperative wound hematoma and one required vascular graft revision. Patients were followed for 4 to 75 months (mean = 23 months). Four patients have required amputations (3 early, 1 late), three of whom had preoperative renal failure. The limb salvage rate has been 72% at 36 months,

The effects of thrombus, thrombectomy and thrombolysis on endothelial function.

OBJECTIVE: this study was undertaken to examine and compare the effects of thrombus, thrombectomy, and thrombolysis on endothelial function as measured by endothelium-dependent vasorelaxation (EDR). METHODS: adult, male New Zealand white rabbits underwent ligation of the left common iliac to femoral artery to induce thrombosis and were then randomly assigned to one of five groups, n=6 in each. Group A consisted of ligation and thrombosis for 4 h. Group B underwent similar ligation for 4 h, but without intraluminal thrombus present. Following 4 h of ligation and thrombosis, Group C underwent thrombectomy while group D was treated with urokinase (UK), 4000 U/min for 30 min. Group E underwent UK infusion alone. The right external iliac artery served as control vessel in each group. All arteries were removed and endothelial function was determined by measuring EDR. RESULTS: the presence of thrombus reduced EDR by 50% (group A) compared to control. Vessels with interrupted flow, but not exposed to thrombus, retained normal EDR (group B). Thrombectomy decreased EDR significantly (group C) compared to thrombolysis (group D) and control. UK did not significantly alter EDR (groups D, E). CONCLUSIONS: exposure of endothelium to thrombus significantly decreases EDR. EDR was not affected by interruption of blood flow in the absence of thrombus. Thrombectomy appeared to cause a further additive insult to the endothelium. In contrast, thrombolysis with UK preserved residual endothelial function. These data suggest that it is important to differentiate the effects of thrombus on endothelium from effects due to thrombectomy or thrombolysis when evaluating treatment modalities for arterial thrombosis.

Diet-induced changes in endothelial-dependent relaxation of the rat aorta.

PURPOSE: Hypertension (HTN), hyperlipidemia (HLP), and hyperinsulinemia are known risk factors for the development of cardiovascular disease. Each has independently been shown to be associated with impaired endothelial function, as demonstrated by decreased endothelial derived relaxation (EDR). Previous work in our laboratory has shown that rats fed a high-fat sucrose (HFS) diet will become insulin resistant, hypertriglyceridemic, and hypertensive. We hypothesize that the development of these diet-induced risk factors is associated with endothelial dysfunction and a significant decrease in EDR. Furthermore, the endothelial dysfunction will be improved by returning to a normal (low-fat complex carbohydrate (LFCC)) diet. METHODS: Adult, male Fischer rats were fed either a LFCC or a HFS diet for 6 months (n = 8 in each group). A third group of rats (SWITCH) was fed a HFS diet for 6 months and then changed to a LFCC diet for 4 weeks. Blood pressure was measured via the tail-cuff method weekly. The rats were sacrificed and aortic ring segments were placed in physiologic tissue baths for measurement of vascular reactivity to various agents. Arterial ring segments were constricted with potassium chloride (K) and phenylephrine (PE). Endothelial-dependent vasorelaxation was measured with acetylcholine (Ach), bradykinin (BK), and calcium ionophore (CA). Endothelial-independent relaxation was measured using sodium nitroprusside (NTP). RESULTS: The HFS diet group developed HTN compared to LFCC group. Vasoconstriction to K and PE were similar in all groups. Vasorelaxation to Ach, BK, and CA was significantly decreased in the HFS group, but returned to baseline in the diet-switched group, as did the systolic blood pressure. There were no differences in relaxation to NTP. CONCLUSIONS: HFS diet-induced HTN is associated with significantly decreased EDR. Switching to a low-fat diet reverses this effect. The vascular smooth muscle contraction and endothelial-independent relaxation are not affected by the diet-induced risk factors. There is a direct and reversible effect of an HFS diet on endothelial function and blood pressure.

Dexamethasone suppresses vascular smooth muscle cell proliferation.

BACKGROUND: Experimental studies in vivo have demonstrated that dexamethasone inhibits neointimal hyperplasia following arterial injury. The mechanisms of this inhibition have not been clearly defined. Our objective was to test the hypothesis that dexamethasone directly suppresses smooth muscle cell (SMC) proliferation by inhibiting cell cycle progression and the expression of key cell cycle-dependent genes. METHODS: Cultured rat aortic SMC were treated with incremental concentrations of dexamethasone and cell number was determined after 72 h. To determine if dexamethasone inhibited cell cycle progression, cells were synchronized, then restimulated to enter the cell cycle, and treated with or without dexamethasone. DNA synthesis was determined 24 h after restimulation by measuring [3H]thymidine incorporation. To define the point of action of dexamethasone in the cell cycle, synchronized SMC were treated with dexamethasone (10(-7) M) at various time points after entry into the cell cycle. Flow cytometry and Northern blots were performed to examine cell cycle progression and the expression of smooth muscle cell cycle-dependent genes c-fos, c-myc, and thymidine kinase (TK). RESULTS: Dexamethasone treatment induced a concentration-dependent inhibition of SMC proliferation and DNA synthesis. The cell cycle progression of synchronized SMC from G1 into S phase was inhibited by dexamethasone, even when added as late as 16 h after restimulation. The expression of TK was suppressed by dexamethasone, while c-fos and c-myc were not affected. CONCLUSIONS: Dexamethasone inhibits the proliferation of SMC in a concentration-dependent fashion. This inhibition is associated with a block in cell cycle progression late in G1 phase of the cell cycle. Consistent with this finding, dexamethasone does not alter the expression of the early cell cycle-dependent genes c-fos and c-myc, but significantly inhibits the expression of TK, a marker of late G1 phase.