Mark D. Iafrati

Select Flavonoids and Whole Juice From Purple Grapes Inhibit Platelet Function and Enhance Nitric Oxide Release

Background—Moderate red wine consumption is inversely associated with coronary ischemia, and both red wine and purple grape juice (PGJ) contain flavonoids with antioxidant and antiplatelet properties believed to be protective against cardiovascular events. Acute cardiac events are also associated with decreased platelet-derived nitric oxide (NO) release. In this study, the effects of PGJ and PGJ-derived flavonoids on platelet function and platelet NO production were determined. Methods and Results—Incubation of platelets with dilute PGJ led to inhibition of aggregation, enhanced release of platelet-derived NO, and decreased superoxide production. To confirm the in vivo relevance of these findings, 20 healthy subjects consumed 7 mL · kg−1 · d−1 of PGJ for 14 days. Platelet aggregation was inhibited after PGJ supplementation, platelet-derived NO production increased from 3.5±1.2 to 6.0±1.5 pmol/108 platelets, and superoxide release decreased from 29.5±5.0 to 19.2±3.1 arbitrary units (P <0.007 and P <0.05, respectively). &agr;-Tocopherol levels increased significantly after PGJ consumption (from 15.6±0.7 to 17.6±0.9 &mgr;mol/L;P <0.009), and the plasma protein–independent antioxidant activity increased by 50.0% (P <0.05). Last, incubation of platelets with select flavonoid fractions isolated from PGJ consistently attenuated superoxide levels but had variable effects on whole-blood aggregation, platelet aggregation, and NO release. Conclusions—Both in vitro incubation and oral supplementation with PGJ decrease platelet aggregation, increase platelet-derived NO release, and decrease superoxide production. These findings may be a result of antioxidant-sparing and/or direct effects of select flavonoids found in PGJ. The suppression of platelet-mediated thrombosis represents a potential mechanism for the beneficial effects of purple grape products, independent of alcohol consumption, in cardiovascular disease.

Stimulation of Toll-Like Receptor 2 in Human Platelets Induces a Thromboinflammatory Response Through Activation of Phosphoinositide 3-Kinase

Cells of the innate immune system use Toll-like receptors (TLRs) to initiate the proinflammatory response to microbial infection. Recent studies have shown acute infections are associated with a transient increase in the risk of vascular thrombotic events. Although platelets play a central role in acute thrombosis and accumulating evidence demonstrates their role in inflammation and innate immunity, investigations into the expression and functionality of platelet TLRs have been limited. In the present study, we demonstrate that human platelets express TLR2, TLR1, and TLR6. Incubation of isolated platelets with Pam3CSK4, a synthetic TLR2/TLR1 agonist, directly induced platelet aggregation and adhesion to collagen. These functional responses were inhibited in TLR2-deficient mice and, in human platelets, by pretreatment with TLR2-blocking antibody. Stimulation of platelet TLR2 also increased P-selectin surface expression, activation of integrin &agr;IIb&bgr;3, generation of reactive oxygen species, and, in human whole blood, formation of platelet–neutrophil heterotypic aggregates. TLR2 stimulation also activated the phosphoinositide 3-kinase (PI3-K)/Akt signaling pathway in platelets, and inhibition of PI3-K significantly reduced Pam3CSK4-induced platelet responses. In vivo challenge with live Porphyromonas gingivalis, a Gram-negative pathogenic bacterium that uses TLR2 for innate immune signaling, also induced significant formation of platelet–neutrophil aggregates in wild-type but not TLR2-deficient mice. Together, these data provide the first demonstration that human platelets express functional TLR2 capable of recognizing bacterial components and activating the platelet thrombotic and/or inflammatory pathways. This work substantiates the role of platelets in the immune and inflammatory response and suggests a mechanism by which bacteria could directly activate platelets.

Duplex assessment of venous reflux and chronic venous insufficiency: The significance of deep venous reflux

PURPOSE This study was undertaken to examine the role of superficial and deep venous reflux, as defined by duplex-derived valve closure times (VCTs), in the pathogenesis of chronic venous insufficiency. METHODS Between January 1992 and November 1995, 320 patients and 500 legs were evaluated with clinical examinations and duplex scans for potential venous reflux. VCTs were obtained with the cuff deflation technique with the patient in the upright position. Imaging was performed at the saphenofemoral junction, the middle segment of the greater saphenous vein, the lesser saphenous vein, the superficial femoral vein, the profunda femoris vein, and the popliteal vein. Not all patients had all segments examined because tests early in the series did not examine the profunda femoris or lesser saphenous vein and because some patients had previous ligation and stripping or venous thrombosis. VCTs were examined for individual segment reflux, grouped into superficial and deep systems, and then correlated with the clinical stage as defined by the SVS/ISCVS original reporting standards in venous disease. Segment reflux was considered present if the VCT was greater than 0.5 seconds, and system reflux was considered present if the sum of the segments was greater than 1.5 seconds. Between-group differences were analyzed with analysis of variance and post hoc tests where appropriate. RESULTS Sixty-nine limbs studied were in class 0, 149 limbs were in class 1, 168 limbs were in class 2, and 114 limbs were in class 3. VCTs in the superficial veins were significantly lower in class 0 than in the other clinical classes. There was no difference in superficial reflux in the symptomatic limbs (classes 1 to 3). Reflux VCTs in the superficial femoral and popliteal veins increased as the clinical symptoms progressed, with a significant increase in class 3 ulcerated limbs when compared with nonuclerated limbs. The incidence of deep venous reflux was 60% in class 3 limbs, compared with 29% in class 2 limbs, whereas the incidence of superficial venous reflux did not differ among the symptomatic limbs. Isolated superficial femoral and popliteal vein reflux was uncommon, even in class 3 limbs, but combined superficial femoral and popliteal vein reflux was found in 53% of class 3 limbs, compared with 18.5% of class 2 limbs. CONCLUSIONS Reflux in the deep venous system plays a significant role in the progression of chronic venous insufficiency. Deep system reflux increases as clinical changes become more severe, with significant axial reflux contributing to ulcer formation.

Relation of Platelet and Leukocyte Inflammatory Transcripts to Body Mass Index in the Framingham Heart Study

Background— Although many genetic epidemiology and biomarker studies have been conducted to examine associations of genetic variants and circulating proteins with cardiovascular disease and risk factors, there has been little study of gene expression or transcriptomics. Quantitative differences in the abundance of transcripts has been demonstrated in malignancies, but gene expression from a large community-based cohort examining risk of cardiovascular disease has never been reported. Methods and Results— On the basis of preliminary microarray data and previously suggested genes from the literature, we measured expression of 48 genes by high-throughput quantitative reverse-transcriptase polymerase chain reaction in 1846 participants of the Framingham Offspring cohort from RNA derived from isolated platelets and leukocytes. A multivariable stepwise regression model was used to assess clinical correlates of quantitative RNA expression. For specific inflammatory platelet-derived transcripts, including ICAM1, IFNG, IL1R1, IL6, MPO, COX2, TNF, TLR2, and TLR4, there were significant associations with higher body mass index (BMI). Compared with platelets, fewer leukocyte-derived transcripts were associated with BMI or other cardiovascular risk factors. Select transcripts were found to be highly heritable, including GPIBA and COX1. Almost uniformly, heritable transcripts were not those associated with BMI. Conclusions— Inflammatory transcripts derived from platelets, particularly those part of the nuclear factor &kgr; B pathway, are associated with BMI, whereas others are heritable. This is the first study, using a large community-based cohort, to demonstrate clinical correlates of gene expression and is consistent with the hypothesis that specific peripheral-blood transcripts play a role in the pathogenesis of coronary heart disease and its risk factors.

Guidelines for the treatment of arterial insufficiency ulcers

1. Co-chaired panel 2. University of Utah, Salt Lake City, UT 3. University of Texas, San Antonio, TX 4. Sinai Hospital/Johns Hopkins Medical Institutions, Baltimore, MD 5. GKT School of Medicine, King’s College, London, UK 6. University of Texas Health Science Center at Houston, TX 7. Sequoia Hospital, Redwood City, CA 8. Maricopa Medical Center, Phoenix, AZ 9. Tufts-New England Medical Center, Boston, MA 10. Southampton University Hospitals Trust NHS, Southampton, UK 11. Penrose–St. Francis Health Services, Colorado Springs, CO 12. Beverly Surgical Associates, Beverly, MA 13. Saint Francis Memorial Hospital, San Francisco, CA 14. Northbay Center for Wound Care, Vacaville, CA, and 15. University of California, San Francisco, CA

The role of amputation as an outcome measure in cellular therapy for critical limb ischemia: implications for clinical trial design

BackgroundAutologous bone marrow-derived stem cells have been ascribed an important therapeutic role in No-Option Critical limb Ischemia (NO-CLI). One primary endpoint for evaluating NO-CLI therapy is major amputation (AMP), which is usually combined with mortality for AMP-free survival (AFS). Only a trial which is double blinded can eliminate physician and patient bias as to the timing and reason for AMP. We examined factors influencing AMP in a prospective double-blinded pilot RCT (2:1 therapy to control) of 48 patients treated with site of service obtained bone marrow cells (BMAC) as well as a systematic review of the literature.MethodsCells were injected intramuscularly in the CLI limbs as either BMAC or placebo (peripheral blood). Six month AMP rates were compared between the two arms. Both patient and treating team were blinded of the assignment in follow-up examinations. A search of the literature identified 9 NO-CLI trials, the control arms of which were used to determine 6 month AMP rates and the influence of tissue loss.ResultsFifteen amputations occurred during the 6 month period, 86.7% of these during the first 4 months. One amputation occurred in a Rutherford 4 patient. The difference in amputation rate between patients with rest pain (5.6%) and those with tissue loss (46.7%), irrespective of treatment group, was significant (p = 0.0029). In patients with tissue loss, treatment with BMAC demonstrated a lower amputation rate than placebo (39.1% vs. 71.4%, p = 0.1337). The Kaplan-Meier time to amputation was longer in the BMAC group than in the placebo group (p = 0.067). Projecting these results to a pivotal trial, a bootstrap simulation model showed significant difference in AFS between BMAC and placebo with a power of 95% for a sample size of 210 patients. Meta-analysis of the literature confirmed a difference in amputation rate between patients with tissue loss and rest pain.ConclusionsBMAC shows promise in improving AMP-free survival if the trends in this pilot study are validated in a larger pivotal trial. The difference in amp rate between Rutherford 4 & 5 patients suggests that these patients should be stratified in future RCTs.

Subfascial endoscopic perforator ligation: An analysis of early clinical outcomes and cost

PURPOSE Early results of subfascial endoscopic perforator surgery (SEPS) were examined. Data on ulcer healing, complications, and costs are presented. METHODS Data were prospectively collected for all patients who underwent SEPS at our institution. A concurrent control group was not available because primary open perforator ligation is no longer performed at our hospital. Preoperative assessment included duplex scanning (valve closure times and perforator mapping), plethysmography, and phlebography. Completeness of therapy was assessed with postoperative duplex mapping of perforating veins. Clinical status was monitored after surgery, and actual costs, including equipment, personnel, and facilities management, are reported. RESULTS Eighteen procedures were performed in 15 patients (mean age, 52 years; range, 42 to 65 years). Two patients underwent bilateral SEPS, and one patient underwent a second procedure on the same leg. Active ulceration (class 6) was present in 14 of 18 limbs (78%), recently healed ulcers (class 5) in two of 18 (11%), and lipodermatosclerosis with edema (class 4) in two. Deep venous insufficiency was present in 14 of 18 (78%). The number of perforating veins ligated per leg ranged from 0 to 12 (mean, 4.3). Follow-up ranged from 3 to 64 weeks (mean, 22 weeks). Complete ulcer healing occurred in eight of 14 limbs (57%) at a mean of 14 weeks. Reduction in ulcer size was noted in four of 14 (29%), and two limbs were not improved. There were no new ulcers. Residual perforating veins were noted in four of 18 limbs. None of the limbs with residual perforating veins had complete healing of ulceration. Operating room costs were higher than those associated with limited-incision open perforator ligation (

Correlation of venous noninvasive tests with the Society for Vascular Surgery/International Society for Cardiovascular Surgery clinical classification of chronic venous insufficiency.

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Validation of Venous Clinical Severity Score (VCSS) with other venous severity assessment tools from the American Venous Forum, National Venous Screening Program

1883). CONCLUSION These preliminary data suggest that when used as part of a treatment plan to correct deep and superficial venous insufficiency SEPS results in a high rate of wound healing, with no recurrent ulceration in this series. Increased operating room costs associated with longer operations and greater disposable expenses will likely be overcome by shortened length of stay and diminished wound complications. These findings emphasize the importance of ligating all incompetent perforating veins, as ulcer healing was never achieved when residual perforating veins were found at follow-up.

Microthrombectomy reduces postsclerotherapy pigmentation: multicenter randomized trial

PURPOSE Noninvasive tests for the evaluation of chronic venous insufficiency (CVI) include quantitative photoplethysmography (QPG), air plethysmography, and duplex ultrasonography measurement of valve closure time (VCT). These tests have been shown to accurately identify the presence of CVI, define the disease, and locate the involved segments. However, the correlation of noninvasive assessment of CVI with the clinical severity (Society for Vascular Surgery/International Society for Cardiovascular Surgery staging) has not been addressed critically. METHOD During an 18-month period, 74 limbs were prospectively evaluated with clinical examination, air plethysmography, QPG and duplex ultrasonography. RESULTS We studied 52 patients with a mean age of 46 years. There were 14 stage 0 limbs, 14 stage 1, 15 stage 2, and 31 stage 3. We found significant differences (p < 0.05) between normal limbs and those with CVI only by VCT and QPG. There were also marked trends toward worsening mean values for reflux (VCT, QPG, and venous filling index) and venous hypertension (residual volume fraction) between stages 0 to 1, and 1 to 2; however, there was a large degree of overlap between all groups. No test discriminated stage 2 from 3. Assessment of calf muscle pump function with ejection fraction showed no difference between any groups. CONCLUSION The Society for Vascular Surgery/International Society for Cardiovascular Surgery criteria for CVI staging distinguishes ulcerated limbs (stage 3) from those with nonulcerating skin changes (hyperpigmentation, brawny edema, and subcutaneous fibrosis) (stage 2). However, we were not able to distinguish these groups by available noninvasive methods. This may imply that these tests are not accurate enough or that the progression from lipodermatosclerosis to frank ulceration is not accounted for by large-vessel hemodynamic changes, but rather by microcirculatory alterations.