Fermin O. Tio

In-stent restenosis: Contributions of inflammatory responses and arterial injury to neointimal hyperplasia

OBJECTIVES We examined the relative contributions of inflammation and arterial injury to neointimal formation in a porcine coronary overstretch restenosis model. BACKGROUND Previous studies established that stents cause neointimal proliferation proportional to injury. Although inflammation has been postulated to be a major contributor to restenosis after angioplasty, there is a paucity of data on the relation between inflammation and subsequent neointimal formation. METHODS Twenty-one pigs underwent balloon injury followed by implantation of oversized, tubular, slotted stents (stent/artery ratio 1.2:1) in the left anterior descending coronary artery. Morphometric analysis of the extent of injury (graded as injury score 0 to 3) and inflammation (graded as inflammation score 0 to 3) 1 month later was assessed and correlated with neointimal formation. RESULTS An inflammatory reaction was observed in 20 of 21 pigs, and significant positive correlations were found between the degree of arterial injury and the extent of the inflammatory reaction (r = 0.80, p < 0.01) and between the extent of inflammatory reaction and the neointimal thickness (r = 0.75, p < 0.01), neointimal area (r = 0.53, p = 0.01) and percent area stenosis (r = 0.66, p < 0.01) within the stents. Importantly, there were areas with inflammation only in the absence of injury, and vice versa, that were also associated with neointimal hyperplasia. CONCLUSIONS These data suggest that the inflammatory reaction plays an equally important role as arterial injury in neointimal formation after coronary stenting, and that anti-inflammatory approaches may be of value to reduce in-stent restenosis.

Transendocardial delivery of autologous Bone marrow enhances collateral perfusion and regional function in pigs with chronic experimental myocardial ischemia

OBJECTIVES We tested the hypothesis that intramyocardial injection of autologous bone marrow (ABM) promotes collateral development in ischemic porcine myocardium. We also defined, in vitro, whether bone marrow (BM) cells secrete vascular endothelial growth factor (VEGF) and macrophage chemoattractant protein-1 (MCP-1). BACKGROUND The natural processes leading to collateral development are extremely complex, requiring multiple growth factors interacting in concert and in sequence. Because optimal angiogenesis may, therefore, require multiple angiogenic factors, we thought that injection of BM, which contains cells that secrete numerous angiogenic factors, might provide optimal therapeutic angiogenesis. METHODS Bone marrow was cultured four weeks in vitro. Conditioned medium was assayed for VEGF and MCP-1 and was added to cultured pig aortic endothelial cells (PAEC) to assess proliferation. Four weeks after left circumflex ameroid implantation, freshly aspirated ABM (n = 7) or heparinized saline (n = 7) was injected transendocardially into the ischemic zone (0.2 ml/injection at 12 sites). Echocardiography to assess myocardial thickening and microspheres to assess perfusion were performed at rest and during stress. RESULTS Vascular endothelial growth factor and MCP-1 concentrations increased in a time-related manner. The conditioned medium enhanced, in a dose-related manner, PAEC proliferation. Collateral flow (ischemic/normal zone X 100) improved in ABM-treated pigs (ABM: 98 +/- 14 vs. 83 +/- 12 at rest, p = 0.001; 89 +/- 18 vs. 78 +/- 12 during adenosine, p = 0.025; controls: 92 +/- 10 vs. 89 +/- 9 at rest, p = 0.49; 78 +/- 11 vs. 77 +/- 5 during adenosine, p = 0.75). Similarly, contractility increased in ABM-treated pigs (ABM: 83 +/- 21 vs. 60 +/- 32 at rest, p = 0.04; 91 +/- 44 vs. 36 +/- 43 during pacing, p = 0.056; controls: 69 +/- 48 vs. 64 +/- 46 at rest, p = 0.74; 65 +/- 56 vs. 37 +/- 56 during pacing, p = 0.23). CONCLUSIONS Bone marrow cells secrete angiogenic factors that induce endothelial cell proliferation and, when injected transendocardially, augment collateral perfusion and myocardial function in ischemic myocardium.

Balloon-expandable intracoronary stents in the adult dog.

We studied the acute and chronic biological reaction to balloon-expandable intracoronary stents in the adult dog. Twenty stainless steel stents were placed, by standard angioplasty techniques, into the left anterior descending, left main, or circumflex coronary arteries of 20 dogs. Angiography was performed at 1, 3, 6, and 12 months and animals were killed in groups of three at 1, 3, 8, and 32 weeks, for gross, light, and electronmicroscopic analysis. All dogs survived, all stents were patent, and there was no evidence of myocardial infarction, spasm, rupture, or aneurysm formation during follow-up (longest 18 months; average, 12 months). The stent was initially covered by a thin layer of thrombus that was replaced later by neointimal muscular proliferation that reached its maximal thickness by 8 weeks (p less than .01). This neointima gradually thinned as it became more sclerotic and less cellular. The stents were covered completely by immature endothelium by 1 week without loss of side branches. We conclude that balloon-expandable intraluminal stents can be safely placed percutaneously into normal canine coronary arteries. Because of rapid endothelialization high patency rates can be expected, thus offering promise for clinical applications in man.

Safety and efficacy of bioabsorbable magnesium alloy stents in porcine coronary arteries

Objective: We aimed to determine the safety and efficacy of biobasorbable magnesium alloy stents in porcine coronary arteries. Bioabsorbable magnesium stents carry the potential to overcome the limitations posed by permanent metallic stents such as chronic inflammation, late stent thrombosis, prolonged antiplatelet therapy, and artifacts when imaged by multislice‐computed tomography or magnetic resonance imaging. Methods: Magnesium alloy stents or stainless steel stents were randomly deployed in coronary arteries of domestic or minipigs. Domestic pigs were sacrificed at 3 days (n = 2) or 28 days, and minipigs at 3 months. Results: At 3 days, magnesium alloy stents were intact, but started to show signs of degradation by 28 days. There was no evidence of stent particle embolization, thrombosis, excess inflammation, or fibrin deposition. At 28 days and 3 months, neointimal area was significantly less in magnesium alloy stent segments (2.44 ± 0.88 mm2 and 1.16 ± 0.19 mm2) as compared with the stainless steel stent segments (5.03 ± 1.5 mm2 and 1.72 ± 0.68 mm2, P < 0.001 and 0.02). Quantitative coronary analysis indicates that percentage area stenosis and percentage diameter stenosis in magnesium alloy stent segments improved significantly at 3 months as compared to 28 days. Despite decreased neointimal hyperplasia, lumen area of the magnesium alloy stented vessels did not improve significantly. Conclusion: Magnesium alloy stents are safe and are associated with less neointima formation; however, reduced neointima did not result in larger lumen.

Local Gene Transfer of phVEGF-2 Plasmid by Gene-Eluting Stents An Alternative Strategy for Inhibition of Restenosis

Background—Drug-eluting stents represent a useful strategy for the prevention of restenosis using various antiproliferative drugs. These strategies share the liability of impairing endothelial recovery, thereby altering the natural biology of the vessel wall and increasing the associated risk of stent thrombosis. Accordingly, we tested the hypothesis that local delivery via gene-eluting stent of naked plasmid DNA encoding for human vascular endothelial growth factor (VEGF)-2 could achieve similar reductions in neointima formation while accelerating, rather than inhibiting, reendothelialization. Methods and Results—phVEGF 2-plasmid (100 or 200 μg per stent)–coated BiodivYsio phosphorylcholine polymer stents versus uncoated stents were deployed in a randomized, blinded fashion in iliac arteries of 40 normocholesterolemic and 16 hypercholesterolemic rabbits. Reendothelialization was nearly complete in the VEGF stent group after 10 days and was significantly greater than in control stents (98.7±1% versus 79.0±6%, P <0.01). At 3 months, intravascular ultrasound analysis revealed that lumen cross-sectional area (4.2±0.4 versus 2.27±0.3 mm2, P <0.001) was significantly greater and percent cross-sectional narrowing was significantly lower (23.4±6 versus 51.2±10, P <0.001) in VEGF stents compared with control stents implanted in hypercholesterolemic rabbits. Transgene expression was detectable in the vessel wall along with improved functional recovery of stented segments, resulting in a 2.4-fold increase in NO production. Conclusions—Acceleration of reendothelialization via VEGF-2 gene–eluting stents provides an alternative treatment strategy for the prevention of restenosis. VEGF-2 gene–eluting stents may be considered as a stand-alone or combination therapy.

Effect of adipose tissue insulin resistance on metabolic parameters and liver histology in obese patients with nonalcoholic fatty liver disease

The role of adipose tissue insulin resistance in the pathogenesis of nonalcoholic fatty liver disease (NAFLD) remains unclear. To evaluate this, we measured in 207 patients with NAFLD (age = 51 ± 1, body mass index = 34.1 ± 0.3 kg/m2) and 22 controls without NAFLD (no NAFLD) adipose tissue insulin resistance by means of a validated index (Adipo‐IRi = plasma free fatty acids [FFA] x insulin [FPI] concentration) and as the suppression of plasma FFA during an oral glucose tolerance test and by a low‐dose insulin infusion. We also explored the relationship between adipose tissue insulin resistance with metabolic and histological parameters by dividing them based on quartiles of adipose tissue insulin resistance (Adipo‐IRi quartiles: Q1 = more sensitive; Q4 = more insulin resistant). Hepatic insulin resistance, measured as an index derived from endogenous glucose production x FPI (HIRi), and muscle insulin sensitivity, were assessed during a euglycemic insulin clamp with 3‐[3H] glucose. Liver fat was measured by magnetic resonance imaging and spectroscopy, and a liver biopsy was performed to assess liver histology. Compared to patients without steatosis, patients with NAFLD were insulin resistant at the level of adipose tissue, liver, and skeletal muscle and had higher plasma aspartate aminotransferase and alanine aminotransferase, triglycerides, and lower high‐density lipoprotein cholesterol and adiponectin levels (all P < 0.01). Metabolic parameters, hepatic insulin resistance, and liver fibrosis (but not necroinflammation) deteriorated as quartiles of adipose tissue insulin resistance worsened (all P < 0.01). Conclusion: Adipose tissue insulin resistance plays a key role in the development of metabolic and histological abnormalities of obese patients with NAFLD. Treatment strategies targeting adipose tissue insulin resistance (e.g., weight loss and thiazolidinediones) may be of value in this population. (HEPATOLOGY 2012)

Passivation of Metallic Stents After Arterial Gene Transfer of phVEGF165Inhibits Thrombus Formation and Intimal Thickening

OBJECTIVES This study sought to test the hypothesis that direct gene transfer of an endothelial cell mitogen could passivate metallic stents by accelerating endothelialization of the prosthesis. BACKGROUND Thrombosis and restenosis comprise the principal clinical manifestations of compromised biocompatibility of endovascular stents. Previous studies have demonstrated that endothelial recovery at sites of balloon injury is a critical determinant of consequent intimal thickening and mural thrombus. We therefore investigated the potential for an endothelial cell mitogen delivered as plasmid DNA to optimize stent biocompatibility. METHODS Naked plasmid DNA encoding vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF) (phVEGF165) was delivered locally using a hydrogel-coated balloon angioplasty catheter to 16 rabbit iliac arteries in which metallic stents had been placed at the site of balloon injury; the contralateral iliac artery of each rabbit was balloon injured and stented but not transfected. RESULTS Stent endothelialization was accelerated by phVEGF165 gene transfer (87.38 +/- 5.06% vs. 33.13 +/- 9.73% [mean +/- SEM] of the planimetered stent surface in the treated vs. contralateral limb, p = 0.005). This was associated with a significant reduction in mural thrombus (3.7 +/- 2.4% vs. 32.7 +/- 9.7%, p = 0.01) at day 7 and intimal thickening (maximal intimal area 0.61 +/- 0.09 vs. 1.44 +/- 0.12 mm2, p < 0.0001) at day 28. No benefit was observed from pCMV-luciferase in 14 similarly instrumented control rabbits. CONCLUSIONS These findings indicate that arterial gene transfer of naked plasmid DNA encoding for an endothelial cell mitogen may successfully passivate endovascular stents by accelerating stent endothelialization, thereby reducing in-stent thrombus and obstruction due to intimal thickening.

Long-Term Pioglitazone Treatment for Patients With Nonalcoholic Steatohepatitis and Prediabetes or Type 2 Diabetes Mellitus: A Randomized Trial

Nonalcoholic fatty liver disease (NAFLD) is reaching epidemic proportions worldwide (1) and is the most common chronic liver condition in obese patients with prediabetes or type 2 diabetes mellitus (T2DM). Histologic findings range from isolated steatosis (with no or minimal inflammation) to severe nonalcoholic steatohepatitis (NASH) and variable perisinusoidal or perivenular fibrosis (2). Patients with T2DM and NASH have the highest risk for cirrhosis and hepatocellular carcinoma (3, 4), and the presence of NAFLD seems to worsen microvascular and macrovascular complications of diabetes (57). Given that most patients with T2DM have NAFLD (812) and many are at risk for NASH even if they have normal liver aminotransferase levels (6, 9, 13, 14), it is surprising that few trials have focused on this population. This distinction (patients with NASH with vs. without T2DM) is relevant because additional metabolic factors, such as hyperglycemia (15, 16), lower adiponectin levels (17, 18), worse dyslipidemia (19, 20), and more severe insulin resistance and hepatic steatosis (10, 16, 1821), may account for the higher rates of severe liver disease observed in patients with T2DM (22). Although the cause of NASH is multifactorial and treatment remains challenging (23), a major factor is the increase in liver triglyceride content caused by chronic release of free fatty acids (FFAs) from insulin-resistant dysfunctional adipose tissue (7, 2427). Because thiazolidinediones target insulin resistance and adipose tissue dysfunction or inflammation that promotes hepatic lipotoxicity in NASH (7, 22, 28) (which is also a prominent feature of T2DM [15]), they may be more helpful for treating steatohepatitis in this population. In predominantly nondiabetic patients with NASH, several studies have reported variable degrees of histologic benefit with thiazolidinediones (2933). In the largest study to date in patients without T2DM (34), pioglitazone was no better than placebo for the primary outcome but was beneficial for secondary outcomes, such as resolution of NASH. However, in patients with prediabetes or T2DM, the only available randomized, controlled trial is a relatively small proof-of-concept study (35). This is disappointing given that there are 29.1 million adults with diabetes (>90% with T2DM) and 86 million with prediabetes (36) in the United States, many of whom are at risk for cirrhosis from NASH. Moreover, because pioglitazone may also halt the progression of prediabetes to T2DM (37), defining its role in patients with prediabetes and NASH is critical. Finally, safety concerns about the long-term use of thiazolidinediones remain (38, 39); therefore, studies with extended thiazolidinedione exposure are needed before a pioglitazone-based approach can be embraced in this population. The aim of our study was to assess the efficacy and safety of long-term pioglitazone treatment in improving liver histologic outcomes in patients with NASH and prediabetes or T2DM. Methods Design Overview This was a single-center, parallel-group, randomized (1:1 allocation), placebo-controlled study, conducted between December 2008 (first patient enrolled) and December 2014 (final data collection). Participants, investigators, and health care providers were blinded to treatment assignment throughout the study. The Institutional Review Board at the University of Texas Health Science Center at San Antonio (UTHSCSA) approved the study, and all participants provided written informed consent before enrollment. In October 2009, while updating registry data for another study, investigators discovered that this trial, which they thought had been registered by other study personnel, was not registered. At the time of registration (ClinicalTrials.gov: NCT00994682), 29 patients (of 97 anticipated) were enrolled in the study. None of these patients had had the follow-up metabolic measurements or liver biopsies (primary outcome) that were to be performed at 18 months, and no interim analyses were done before the trial was registered. A recent review of ClinicalTrials.gov (November 2015) revealed that the initial trial registration data erroneously stated that patients with normal glucose tolerance would be randomly assigned to treatment or placebo. Given that the trials eligibility criteria required patients to have an abnormal oral glucose tolerance test (OGTT) result (that is, prediabetes or T2DM), the investigators never planned to enroll patients with normal glucose tolerance. This error in trial registration was corrected by the principal investigator. The trial registry states that the primary end point is liver histologic outcomes (Kleiner criteria [40]) at 18 months, and these data are presented in Appendix Table 1. In this article, the primary end point is defined as a reduction of at least 2 points in 2 categories of the NAFLD activity score (NAS) without worsening of fibrosis, an outcome that was not specified in the original registration. This end point has been accepted by investigators in this field as representing significant change in liver histologic outcomes in clinical trials involving patients with NASH (34, 4143). Some secondary outcomes that were assessed, such as insulin secretion, prevention of the onset of T2DM or reversal of glucose intolerance, measurement of visceral fat by magnetic resonance imaging, bone density measurement via dual-energy x-ray absorptiometry (DXA), plasma measurements of bone metabolism, and molecular metabolic pathways, are not reported in this article. Appendix Table 1. Liver Histologic Variables at Baseline and After 18 mo, Based on Observed Data* Setting and Participants Participants were recruited from the general population of San Antonio, Texas, via newspaper advertisements and from the endocrinology and hepatology clinics at UTHSCSA and the Veterans Affairs Medical Center. Persons were eligible for the trial if they had histologically confirmed NASH and either prediabetes or T2DM. All patients had a screening 2-hour OGTT to diagnose or confirm a diagnosis of prediabetes or T2DM. Prediabetes was defined as impaired fasting glucose (5.6 to 6.9 mmol/L [100 to 125 mg/dL]), impaired glucose tolerance (7.8 to 11.1 mmol/L [140 to 199 mg/dL] on an OGTT), or a hemoglobin A1c level of 5.7% to 6.4%. Exclusion criteria included use of thiazolidinediones or vitamin E; other causes of liver disease (22) or abnormal laboratory results (such as an aspartate aminotransferase [AST] or alanine aminotransferase [ALT] level 3 times the upper limit of normal [ULN]); type 1 diabetes mellitus; or severe heart, hepatic, or renal disease. Detailed inclusion and exclusion criteria are provided in the Appendix. Randomization and Interventions After initial screening (medical history, physical examination, laboratory tests, and 75-g OGTT), patients began receiving placebo and were instructed by the research dietician (C.D.) to keep physical activity and diet constant during the run-in phase (mean duration, 1 month). After completion of baseline metabolic measurements, participants were prescribed a hypocaloric diet (500kcal/d deficit from the calculated weight-maintaining diet) and were randomly assigned in a 1:1 ratio to either pioglitazone (Actos [Takeda Pharmaceuticals]), 30 mg/d (titrated after 2 months to 45 mg/d), or placebo. Randomization (computer-generated) and patient allocation were performed by the research pharmacist without stratification and using a block factor of 4, which was unknown to investigators. Takeda Pharmaceuticals provided pioglitazone and placebo pills with identical physical characteristics, which were stored at the research pharmacy and dispensed in identical bottles. Outcomes and Follow-up The primary outcome was a reduction of at least 2 points in 2 histologic categories of the NAS without worsening of fibrosis after 18 months of therapy. Secondary liver histologic outcomes included resolution of NASH; improvement in individual histologic scores; or improvement in a combined histologic outcome, defined as a reduction in ballooning with at least a 2-point improvement in the NAS or an absolute NAS of 3 or lower (with improvement in steatosis or inflammation) without worsening of fibrosis. Baseline liver biopsy specimens were read by a team of experienced clinical pathologists to establish or rule out the presence of NASH and thus determine whether patients were included or excluded. At the end of the study, all biopsy specimens were reread by an experienced research pathologist (F.T.), who was blinded to patient identity, intervention assignment, and pretreatment or posttreatment sequence (0, 18, or 36 months). Biopsy specimens were read by the research pathologist 2 times, with good to excellent intraobserver variability (agreement >75% for all histologic parameters). Diagnosis of definite NASH was defined as zone 3 accentuation of macrovesicular steatosis (any grade), hepatocellular ballooning (any degree), and lobular inflammatory infiltrates (any amount). The NAS was calculated as the sum of the steatosis, inflammation, and ballooning grades from the liver biopsy, and histopathologic changes were determined by using standard criteria (44). Additional secondary outcomes included the following: 1) fasting plasma glucose, fasting plasma insulin, FFA, hemoglobin A1c, fasting plasma lipid profile, adiponectin, and cytokeratin-18 concentrations; 2) total body fat percentage, measured by DXA; 3) hepatic triglyceride content, measured by magnetic resonance and proton spectroscopy (1H-MRS) as previously described (14, 16, 35, 45) (baseline and 18 months only); 4) glucose tolerance and insulin secretion on an OGTT; 5) endogenous glucose production (EGP), rate of glucose disappearance (R d), and insulin-induced suppression of EGP and plasma FFA concentration, all measured during a euglycemic insulin clamp with tritiated glucose and indirect calorimetry (baseline and 18 months only) as previously reported (16

In Vivo Comparison Between Optical Coherence Tomography and Intravascular Ultrasound for Detecting Small Degrees of In-Stent Neointima After Stent Implantation

OBJECTIVES The purpose of this study was to evaluate optical coherence tomography (OCT) for detecting small degrees of in-stent neointima (ISN) after stent implantation compared with intravascular ultrasound (IVUS). BACKGROUND The importance of detecting neointimal coverage of stent struts has grown with the appreciation of the increased risk for late stent thrombosis after drug-eluting stent (DES) implantation. Intravascular ultrasound, the current standard for evaluating the status of DES, lacks the resolution to detect the initial neointimal coverage. Optical coherence tomography has greater resolution but has not yet been compared with IVUS in vivo with histological correlation for validation. METHODS Intravascular ultrasound and OCT were performed with motorized pullback imaging in 6 pigs across 33 stents, 1 month after implantation. Each pig was euthanized, and histological measurements of vessel, stent, and lumen dimensions were performed in 3 sections of each stent. A small degree of ISN was defined as occupying <30% of the stent area measured with histology. The IVUS, OCT, and histological assessment of ISN were compared in matched cross-sections of the stents with a small degree of ISN. RESULTS Eleven stents had a small degree of ISN (average ISN area: 1.26 +/- 0.46 mm(2), and percent area obstruction: 21.4 +/- 5.2%). Compared with histology, the diagnostic accuracy of OCT (area under the receiver operating characteristic curve [AUC] = 0.967, 95% confidence interval [CI] 0.914 to 1.019) was higher than that of IVUS (AUC = 0.781, 95% CI 0.621 to 0.838). CONCLUSIONS Optical coherence tomography detects smaller degrees of ISN more accurately than IVUS and might be a useful method for identifying neointimal coverage of stent struts after DES implantation.

Limited value of plasma cytokeratin-18 as a biomarker for NASH and fibrosis in patients with non-alcoholic fatty liver disease

BACKGROUND & AIMS Liver biopsy is the only reliable way of diagnosing and staging NASH but its invasive nature limits its use. Plasma caspase-generated cytokeratin-18 fragments (CK-18) have been proposed as a non-invasive alternative. We studied its clinical value in a large multiethnic NAFLD population and examined its relationship to clinical/metabolic/histological parameters. METHODS 424 middle-aged subjects in whom we measured adipose tissue, liver and muscle insulin resistance (IR), liver fat by MRS (n=275) and histology (n=318). RESULTS Median CK-18 were elevated in patients with vs. without NAFLD by MRS (209 [IQR: 137-329] vs. 122 [IQR: 98-155]U/L) or with vs. without NASH (232 [IQR: 151-387] vs. 170 [IQR: 135-234]U/L, both p<0.001). Plasma CK-18 raised significantly with any increase in steatosis, inflammation and fibrosis, but there was a significant overlap across disease severity. The CK-18 AUROC to predict NAFLD, NASH or fibrosis were 0.77 (95% CI=0.71-0.84), 0.65 (95% CI=0.59-0.71) and 0.68 (95% CI=0.61-0.75), respectively. The overall sensitivity/specificity for NAFLD, NASH and fibrosis were 63% (57-70%)/83% (69-92%), 58% (51-65%)/68% (59-76%) and 54% (44-63%)/85% (75-92%), respectively. CK-18 correlated most strongly with ALT (r=0.57, p<0.0001) and adipose tissue IR (insulin-suppression of FFA: r=-0.43; p<0.001), less with steatosis, lobular inflammation and fibrosis (r=0.28-0.34, all p<0.001), but not with ballooning, BMI, metabolic syndrome or T2DM. CONCLUSIONS Plasma CK-18 has a high specificity for NAFLD and fibrosis, but its limited sensitivity makes it inadequate as a screening test for staging NASH. Whether combined as a diagnostic panel with other biomarkers or clinical/laboratory tests may prove useful requires further study.