M. Ng

Strikingly different angiogenic properties of endothelial progenitor cell subpopulations: insights from a novel human angiogenesis assay.

OBJECTIVES An endothelial cell (EC)-specific angiogenesis assay was developed to functionally characterize angiogenic properties of 2 distinct putative endothelial progenitor cells (EPCs): early EPCs and late outgrowth endothelial cells (OECs). BACKGROUND Endothelial progenitor cells promote revascularization of ischemic tissue. However, the nature of different EPCs and their role in angiogenesis remains debated. METHODS Tubulogenesis was assessed by immunohistochemistry in co-cultures of differentiated ECs (including human umbilical vein, coronary artery, and microvascular ECs) or non-ECs with monolayers of human fibroblasts (MRC5). Using adaptations of the co-culture assay, early EPCs and OECs, isolated from peripheral blood mononuclear cells, were assessed by 3-dimensional immunofluorescence microscopy for their capacity for: 1) independent tubulogenesis; 2) incorporation into pre-existing vascular networks; and 3) paracrine angiogenic effects using transwell cultures. RESULTS Branched interconnecting EC-specific tubules formed with all differentiated ECs after 72 h. Proangiogenic and antiangiogenic agents modulated tubulogenesis appropriately (vascular endothelial growth factor 10 ng: +142 +/- 13%, 1 microM anti-vascular endothelial growth factor: -44 +/- 7% vs. control, p < 0.001). In contrast, early EPCs, along with nonendothelial cell types, failed to independently form tubules or incorporate into differentiated EC tubules. Nevertheless, early EPCs indirectly augmented tubulogenesis by differentiated ECs even when physically separated by transwells (+115 +/- 4% vs. control; p < 0.001). By contrast, OECs independently formed tubules and incorporated into differentiated EC tubules but exerted no significant paracrine angiogenic effects. CONCLUSIONS A novel EC-specific tubulogenesis assay highlights strikingly different angiogenic properties of different EPCs: late OECs directly participate in tubulogenesis, whereas early EPCs augment angiogenesis in a paracrine fashion, with implications for optimizing cell therapies for neovascularization.

Invasive Assessment of the Coronary Microcirculation Superior Reproducibility and Less Hemodynamic Dependence of Index of Microcirculatory Resistance Compared With Coronary Flow Reserve

Background— A simple, reproducible invasive method for assessing the coronary microcirculation is lacking. A novel index of microcirculatory resistance (IMR) has been shown in animals to correlate with true microvascular resistance and, unlike coronary flow reserve (CFR), to be independent of the epicardial artery. We sought to compare the reproducibility and hemodynamic dependence of IMR with CFR in humans. Methods and Results— Using a pressure-temperature sensor-tipped coronary wire, thermodilution-derived CFR and IMR were measured, along with fractional flow reserve (FFR), in 15 coronary arteries (15 patients) under the following hemodynamic conditions: (1) twice at baseline; (2) during right ventricular pacing at 110 bpm; (3) during intravenous infusion of nitroprusside; and (4) during intravenous dobutamine infusion. Mean CFR did not change during baseline measurements or during nitroprusside infusion but decreased during pacing (from 3.1±1.1 at baseline to 2.3±1.2 during pacing, P<0.05) and during dobutamine infusion (from 3.0±1.0 to 1.7±0.6 with dobutamine, P<0.0001). By comparison, mean values for IMR and FFR remained similar throughout all hemodynamic conditions. The mean coefficient of variation between 2 baseline measurements was significantly lower for IMR (6.9±6.5%) and FFR (1.6±1.6%) than for CFR (18.6±9.6%; P<0.01). Mean correlation between baseline measurements and each hemodynamic intervention was superior for IMR (r=0.90±0.05) and FFR (r=0.86±0.12) compared with CFR (r=0.70±0.05; P<0.05). Conclusions— Compared with CFR, IMR provides a more reproducible assessment of the microcirculation, which is independent of hemodynamic perturbations. Simultaneous measurement of FFR and IMR may provide a comprehensive and specific assessment of coronary physiology at both epicardial and microvascular levels, respectively.

Predictive Value of the Index of Microcirculatory Resistance in Patients With ST-Segment Elevation Myocardial Infarction

OBJECTIVES The objective of this study is to evaluate the predictive value of the index of microcirculatory resistance (IMR) in patients undergoing primary percutaneous coronary intervention (PCI) for ST-segment elevation myocardial infarction (STEMI). BACKGROUND Despite adequate epicardial artery reperfusion, a number of patients with STEMI have a poor prognosis because of microvascular damage. Assessing the status of the microvasculature in this setting remains challenging. METHODS In 29 patients after primary PCI for STEMI, IMR was measured with a pressure sensor/thermistor-tipped guidewire. The Thrombolysis In Myocardial Infarction (TIMI) myocardial perfusion grade, TIMI frame count, coronary flow reserve, and ST-segment resolution were also recorded. RESULTS The IMR correlated significantly with the peak creatinine kinase (CK) (R = 0.61, p = 0.0005) while the other measures of microvascular dysfunction did not. In patients with an IMR greater than the median value of 32 U, the peak CK was significantly higher compared with those having values <or=32 U (3,128 +/- 1,634 ng/ml vs. 1,201 +/- 911 ng/ml, p = 0.002). The IMR correlated significantly with 3-month echocardiographic wall motion score (WMS) (R = 0.59, p = 0.002) while the other measures of microvascular function did not. The WMS at 3-month follow-up was significantly worse in the group with an IMR >32 U compared with <or=32 U (28 +/- 7 vs. 20 +/- 4, p = 0.001). On multivariate analysis, IMR was the strongest predictor of peak CK and 3-month WMS. The IMR was the only significant predictor of recovery of left ventricular function on the basis of the percent change in WMS (R = 0.50, p < 0.01). CONCLUSIONS Compared to standard measures, IMR appears to be a better predictor of microvascular damage after STEMI, both acutely and in short term follow-up.

A multilayered synthetic human elastin/polycaprolactone hybrid vascular graft with tailored mechanical properties.

Small-diameter synthetic vascular graft materials fail to match the patency of human tissue conduits used in vascular bypass surgery. The foreign surface retards endothelialization and is highly thrombogenic, while the mismatch in mechanical properties induces intimal hyperplasia. Using recombinant human tropoelastin, we have developed a synthetic vascular conduit for small-diameter applications. We show that tropoelastin enhances endothelial cell attachment (threefold vs. control) and proliferation by 54.7 ± 1.1% (3 days vs. control). Tropoelastin, when presented as a monomer and when cross-linked into synthetic elastin for biomaterials applications, had low thrombogenicity. Activation of the intrinsic pathway of coagulation, measured by plasma clotting time, was reduced for tropoelastin (60.4 ± 8.2% vs. control). Platelet attachment was also reduced compared to collagen. Reductions in platelet interactions were mirrored on cross-linked synthetic elastin scaffolds. Tropoelastin was subsequently incorporated into a synthetic elastin/polycaprolactone conduit with mechanical properties optimized to mimic the human internal mammary artery, including permeability, compliance, elastic modulus and burst pressure. Further, this multilayered conduit presented a synthetic elastin internal lamina to circulating blood and demonstrated suturability and mechanical durability in a small scale rabbit carotid interposition model.

Covalent immobilisation of tropoelastin on a plasma deposited interface for enhancement of endothelialisation on metal surfaces

Currently available endovascular metallic implants such as stents exhibit suboptimal biocompatibility in that they re-endothelialise poorly leaving them susceptible to thrombosis. To improve the interaction of these implants with endothelial cells we developed a surface coating technology, enabling the covalent attachment of biomolecules to previously inert metal surfaces. Using horseradish peroxidase as a probe, we demonstrate that the polymerised surface can retain the presentation and activity of an immobilised protein. We further demonstrated the attachment of tropoelastin, an extracellular matrix protein critical to the correct arrangement and function of vasculature. Not only it is structurally important, but it plays a major role in supporting endothelial cell growth, while modulating smooth muscle cell infiltration. Tropoelastin was shown to bind to the surface in a covalent monolayer, supplemented with additional physisorbed multilayers on extended incubation. The physisorbed tropoelastin layers can be washed away in buffer or SDS while the first layer of tropoelastin remains tightly bound. The plasma coated stainless steel surface with immobilised tropoelastin was subsequently found to have improved biocompatibility by promoting endothelial cell attachment and proliferation relative to uncoated stainless steel controls. Tropoelastin coatings applied to otherwise inert substrates using this technology could thus have broad applications to a range of non-polymeric vascular devices.

Three-dimensional and two-dimensional quantitative coronary angiography, and their prediction of reduced fractional flow reserve

AIMS We investigated whether three-dimensional (3D) and two-dimensional quantitative coronary angiography (2D-QCA) measurements differed in their accuracy in predicting reduced fractional flow reserve (FFR), and how this varied with stenosis severity and the FFR cut-off used. METHODS AND RESULTS Three-dimensional and 2D-QCA were compared in their measurements of minimum luminal area (MLA), percentage area stenosis, lesion length, minimum luminal diameter (MLD) and percentage diameter stenosis, and in their prediction of functionally significant FFR. In total, 63 target lesions were interrogated in 63 patients undergoing elective percutaneous coronary intervention. Of all measurements of lesion severity obtained by 3D-QCA, MLA best correlated with FFR (R = 0.63, P < 0.001), and was the most accurate predictor of FFR < 0.75 (C statistic 0.86, P < 0.001). Of 2D-QCA measurements, MLD correlated best with FFR (R = 0.58, P < 0.001), and best predicted FFR < 0.75 (C statistic 0.80, P < 0.001). Overall, 3D-QCA showed a non-significant trend towards more accurate prediction of FFR than 2D-QCA, especially in intermediate lesions. The relationship between FFR and apparent stenosis severity was found to be curvilinear. Both 3D- and 2D-QCA were less accurate in intermediate lesions, and in predicting FFR ≤ 0.80 than in predicting FFR <0.75. CONCLUSIONS The accuracy of QCA in predicting functionally significant FFR is limited and is dependent on FFR cut-off used and lesion severity. Where FFR is not available or contraindicated, 3D-QCA may assist in the evaluation of coronary lesions of intermediate severity.

Transcatheter aortic valve implantation for high-risk patients with severe aortic stenosis: A systematic review.

OBJECTIVES The present systematic review objectively assessed the safety and clinical effectiveness of transcatheter aortic valve implantation for patients at high surgical risk with severe aortic stenosis. METHODS Electronic searches were performed in 6 databases from January 2000 to March 2009. The end points included feasibility, safety, efficacy, and durability. Clinical effectiveness was synthesized through a narrative review with full tabulation of results of all included studies. RESULTS The current evidence on transcatheter aortic valve implantation for aortic stenosis is limited to short-term observational studies. The overall procedural success rates ranged from 74% to 100%. The incidence of major adverse events included 30-day mortality (0%-25%), major ventricular tachyarrhythmia (0%-4%), myocardial infarction (0%-15%), cardiac tamponade (2%-10%), stroke (0%-10%), conversion to surgery (0%-8%), moderate to major paravalvular leak (4%-35%), vascular complication (8%-17%), valve-in-valve procedure (2%-12%), and aortic dissection/perforation (0%-4%). The overall 30-day major adverse cardiovascular and cerebral events ranged from 3% to 35%. The mean aortic valve area ranged from 0.5 to 0.8 cm(2) before and 1.3 to 2.0 cm(2) after transcatheter aortic valve implantation. The mean pressure gradient ranged from 34 to 58 mm Hg before and 3 to 12 mm Hg after transcatheter aortic valve implantation. There was no significant deterioration in echocardiography measurements during the assessment period. Death rate at 6 months postprocedure ranged from 18% to 48%. No studies had adequate follow-up to reliably evaluate long-term outcomes. CONCLUSIONS The procedure has a potential for serious complications. Although short-term efficacy based on echocardiography measurements is good, there is little evidence on long-term outcomes. The use of transcatheter aortic valve implantation should be considered only within the boundaries of clinical trials.

Cell therapies for therapeutic angiogenesis: back to the bench

Abstract The discovery, over a decade ago, of endothelial progenitor cells that are able to participate in neovascularization of adult tissue has been greeted enthusiastically because of the potential for new cell-based therapies for therapeutic angiogenesis. Since that time, an ever-growing list of candidate cells has been proposed for cardiovascular regeneration. However, to date, pre-clinical and clinical studies evaluating the therapeutic potential of various cell therapies have reported conflicting results, generating controversy. Key issues within the field of cell therapy research include a lack of uniform cellular definitions, as well as inadequate functional characterization of the role of putative stem/progenitor cells in angiogenesis. Given the mixed results of initial clinical studies, there is now a scientific imperative to understand better the vascular biology of candidate cells in order to better translate cell therapy to the bedside. This review will provide a translationally relevant overview of the biology of candidate stem/progenitor cells for therapeutic angiogenesis.

The immobilization of recombinant human tropoelastin on metals using a plasma-activated coating to improve the biocompatibility of coronary stents

Current endovascular stents have sub-optimal biocompatibility reducing their clinical efficacy. We previously demonstrated a plasma-activated coating (PAC) that covalently bound recombinant human tropoelastin (TE), a major regulator of vascular cells in vivo, to enhance endothelial cell interactions. We sought to develop this coating to enhance its mechanical properties and hemocompatibility for application onto coronary stents. The plasma vapor composition was altered by incorporating argon, nitrogen, hydrogen or oxygen to modulate coating properties. Coatings were characterized for 1) surface properties, 2) mechanical durability, 3) covalent protein binding, 4) endothelial cell interactions and 5) thrombogenicity. The N(2)/Ar PAC had optimal mechanical properties and did not delaminate after stent expansion. The N(2)/Ar PAC was mildly hydrophilic and covalently bound the highest proportion of TE, which enhanced endothelial cell proliferation. Acute thrombogenicity was assessed in a modified Chandler loop using human blood. Strikingly, the N(2)/Ar PAC alone reduced thrombus weight by ten-fold compared to 316L SS, a finding unaltered with immobilized TE. Serum soluble P-selectin was reduced on N(2)/Ar PAC and N(2)/Ar PAC + TE (p < 0.05), consistent with reduced platelet activation. We have demonstrated a coating for metal alloys with multifaceted biocompatibility that resists delamination and is non-thrombogenic, with implications for improving coronary stent efficacy.

Animal models for the assessment of novel vascular conduits

The development of an ideal small-diameter conduit for use in vascular bypass surgery has yet to be achieved. The ongoing innovation in biomaterial design generates novel conduits that require preclinical assessment in vivo, and a number of animal models have been used for this purpose. This article examines the rationale behind animal models used in the assessment of small-diameter vascular conduits encompassing the commonly used species: baboons, sheep, pigs, dogs, rabbits, and rodents. Studies on the comparative hematology for these species relative to humans are summarized, and the hydrodynamic values for common implant locations are also compared. The large- and small-animal models are then explored, highlighting the characteristics of each that determine their relative utility in the assessment of vascular conduits. Where possible, the performance of expanded polytetrafluoroethylene is given in each animal and in each location to allow direct comparisons between species. New challenges in animal modeling are outlined for the assessment of tissue-engineered graft designs. Finally, recommendations are given for the selection of animal models for the assessment of future vascular conduits.