Farhad Forudi

Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy

BACKGROUND Myocardial regeneration via stem-cell mobilisation at the time of myocardial infarction is known to occur, although the mechanism for stem-cell homing to infarcted tissue subsequently and whether this approach can be used for treatment of ischaemic cardiomyopathy are unknown. We investigated these issues in a Lewis rat model (ligation of the left anterior descending artery) of ischaemic cardiomyopathy. METHODS We studied the effects of stem-cell mobilisation by use of granulocyte colony-stimulating factor (filgrastim) with or without transplantation of syngeneic cells. Shortening fraction and myocardial strain by tissue doppler imaging were quantified by echocardiography. FINDINGS Stem-cell mobilisation with filgrastim alone did not lead to engraftment of bone-marrow-derived cells. Stromal-cell-derived factor 1 (SDF-1), required for stem-cell homing to bone marrow, was upregulated immediately after myocardial infarction and downregulated within 7 days. 8 weeks after myocardial infarction, transplantation into the peri-infarct zone of syngeneic cardiac fibroblasts stably transfected to express SDF-1 induced homing of CD117-positive stem cells to injured myocardium after filgrastim administration (control vs SDF-1-expressing cardiac fibroblasts mean 7.2 [SD 3.4] vs 33.2 [6.0] cells/mm2, n=4 per group, p<0.02) resulting in greater left-ventricular mass (1.24 [0.29] vs 1.57 [0.27] g) and better cardiac function (shortening fraction 9.2 [4.9] vs 17.2 [4.2]%, n=8 per group, p<0.05). INTERPRETATION These findings show that SDF-1 is sufficient to induce therapeutic stem-cell homing to injured myocardium and suggest a strategy for directed stem-cell engraftment into injured tissues. Our findings also indicate that therapeutic strategies focused on stem-cell mobilisation for regeneration of myocardial tissue must be initiated within days of myocardial infarction unless signalling for stem-cell homing is re-established.

Influence of Blockade at Specific Levels of the Coagulation Cascade on Restenosis in a Rabbit Atherosclerotic Femoral Artery Injury Model

BACKGROUND The relation among the coagulation cascade, its individual proteins, and the response to vascular injury is largely undefined. We have evaluated the effect of four probes that block specific levels of coagulation cascade on neointimal hyperplasia in the atherosclerotic rabbit arterial injury model. METHODS AND RESULTS Focal femoral atherosclerosis was induced by air-desiccation injury and hypercholesterolemic diet in 48 New Zealand White rabbits, followed by balloon angioplasty. Active-site inactivated factor VIIa (DEGR-VIIa), which blocks the binding of factor VIIa to tissue factor, was administered (n = 12 arteries) by intravenous bolus (1 mg/kg) at the time of balloon angioplasty and followed by infusion of 50 micrograms.kg-1.h-1 for 3 days; for the control (n = 13 arteries), 150 U heparin was injected as bolus and followed by infusion of saline at 50 microL.kg-1.min-1. Recombinant tissue factor pathway inhibitor (TFPI), which binds factor Xa and inhibits the tissue factor-factor VIIa complex and factor Xa, was given as a 1 mg/kg bolus followed by 15 micrograms.kg-1.min-1 infusion for 3 days (n = 17 arteries). Recombinant tick anticoagulant peptide (TAP; n = 15 arteries) and hirudin (n = 14 arteries), which block factor Xa and thrombin, respectively, were administered as a 1 mg/kg bolus followed by 5 micrograms.kg-1.min-1 infusion for 3 days. These three groups had their own controls (n = 14 arteries). There were no differences among treatment groups in preangioplasty and postangioplasty minimal luminal diameter (MLD) by angiography. The mean MLD 21 days after balloon angioplasty was significantly different between control and DEGR-VIIa-treated groups (0.74 +/- 0.25 and 1.24 +/- 0.27 mm, respectively; P = .0001) and between the TFPI-treated group and others (0.88 +/- 0.21 mm for control, 0.97 +/- 0.22 mm for hirudin-treated, 0.98 +/- 0.14 mm for TAP-treated, and 1.32 +/- 0.21 mm for TFPI-treated arteries; P = .0001 by ANOVA). By quantitative histological analysis, the ratio of neointimal cross-sectional area compared with the area of internal elastic lamina in the DEGR-VIIa-treated group was significantly less than control (0.48 +/- 0.12 versus 0.67 +/- 0.12, P = .0001), and the ratio of neointimal cross-sectional area to the area demarcated by the internal elastic lamina of the TFPI-treated group was significantly reduced compared with the other groups (0.46 +/- 0.20 for TFPI-treated, 0.67 +/- 0.15 for hirudin-treated, 0.61 +/- 0.15 for TAP-treated, and 0.64 +/- 0.13 for control groups; P = .003). CONCLUSIONS Treatment with DEGR-VIIa or TFPI for 3 days in this rabbit atherosclerotic injury model reduced angiographic restenosis and decreased neointimal hyperplasia compared with controls. These findings highlight the importance of early initiators of the extrinsic coagulation pathway, especially factor VII and tissue factor, in the response to arterial injury.

Receptor for AGE (RAGE) Mediates Neointimal Formation in Response to Arterial Injury

Background—Receptor for advanced-glycation end products (RAGE) and its ligands AGEs and S100/calgranulins have been implicated in a range of disorders. However, the role of RAGE/ligand interaction in neointimal hyperplasia after vascular injury remains unclear. Methods and Results—We examined the expression of RAGE and its ligands after balloon injury of the carotid artery in both Zucker diabetic and nondiabetic rats. Using a soluble portion of the extracellular domain of RAGE, we determined the effects of suppressing RAGE/ligand interaction on vascular smooth muscle cell (VSMC) proliferation and neointimal formation after arterial injury. We demonstrate a significantly increased accumulation of AGE and immunoreactivities of RAGE and S100/calgranulins in response to balloon injury in diabetic compared with nondiabetic rats. Blockade of RAGE/ligand interaction significantly decreased S100-stimulated VSMC proliferation in vitro and bromodeoxyuridine (BrdU)–labeled proliferating VSMC in vivo, and suppressed neointimal formation and increased luminal area in both Zucker diabetic and nondiabetic rats. Conclusions—These findings indicate that RAGE/ligand interaction plays a key role in neointimal formation after vascular injury irrespective of diabetes status and suggest a novel target to minimize neointimal hyperplasia.

Increased Expression of Thrombospondin-1 in Vessel Wall of Diabetic Zucker Rat

Background—Thrombospondin-1 (TSP-1) expression in the vascular wall has been related to the development of atherosclerotic lesions and restenosis. TSP-1 promotes the development of neointima and has recently been associated with atherogenesis at a genetic level. Because TSP-1 expression is responsive to glucose stimulation in mesangial cells, we hypothesized that glucose may stimulate its production by vascular cells. Thus, TSP-1 expression in the blood vessel wall may increase, providing a molecular link between diabetes and accelerated vascular lesion development. Methods and Results—To determine whether the expression level of TSP-1 in vessel wall is increased in diabetes, aorta and carotid arteries of Zucker rats were used for immunostaining, Western blotting, and in situ RNA hybridization. A significant increase in TSP-1 expression was found in the adventitia of blood vessels from diabetic rats. Consistent with the well-known antiangiogenic effect of TSP-1, the number of vasa vasorum was reduced in aortas from diabetic rats. In cultured endothelial cells, vascular smooth muscle cells, and fibroblasts, TSP-1 expression increased in response to glucose stimulation (>30-fold). After balloon catheter injury to carotid arteries, expression of TSP-1 protein and mRNA was higher at all time points in the vessels of diabetic rats. Conclusions—Increased expression of TSP-1 in blood vessels in diabetes may represent a new link between diabetes, atherogenesis, and accelerated restenosis. This increase in TSP-1 production may be a direct response of vascular cells to glucose.

Direct delivery of syngeneic and allogeneic large-scale expanded multipotent adult progenitor cells improves cardiac function after myocardial infarct

BACKGROUND Multipotent adult progenitor cells (MAPC) comprise interesting candidates for myocardial regeneration because of a broad differentiation ability and immune privilege. We aimed to compare the improvement of cardiac function by syngeneic and allogeneic MAPC produced on a large scale using a platform optimized from MAPC research protocols. METHODS Myocardial infarction was induced in Lewis rats by direct left anterior descending ligation followed immediately by direct injection into the infarct border zone of either Sprague-Dawley or Lewis MAPC from large-scale expansions. Echocardiography was performed to evaluate improvement in cardiac function, and immunohistochemistry was performed to identify MAPC within the infarct zone. RESULTS Significant increases were observed in functional performance in animals transplanted with expanded MAPC compared with saline controls, with no significant differences between the syngeneic and allogeneic groups. Immunostaining demonstrated significant engraftment of expanded MAPC at 1 day after acute myocardial infarction, with <10% of either syngeneic or allogeneic cells remaining at 6 weeks. At this point there was no evidence of myocardial regeneration. However, a significant increase in vascular density within the infarct zone in MAPC-transplanted animals was observed, and MAPC were found to produce high levels of VEGF in culture. DISCUSSION These findings support a model in which delivery of expanded MAPC following acute myocardial infarction results in improvement in cardiac function because of paracrine effects resulting in vascular density increases, as well as potentially other trophic effects, supporting newly injured cardiac myocytes. Thus transplantation with MAPC may represent a promising therapeutic strategy with application in the stimulation of neovascularization in ischemic heart disease.

Disruption of Protein Kinase A Interaction with A-kinase-anchoring Proteins in the Heart in Vivo EFFECTS ON CARDIAC CONTRACTILITY, PROTEIN KINASE A PHOSPHORYLATION, AND TROPONIN I PROTEOLYSIS

Protein kinase A (PKA)-dependent phosphorylation is regulated by targeting of PKA to its substrate as a result of binding of regulatory subunit, R, to A-kinase-anchoring proteins (AKAPs). We investigated the effects of disrupting PKA targeting to AKAPs in the heart by expressing the 24-amino acid regulatory subunit RII-binding peptide, Ht31, its inactive analog, Ht31P, or enhanced green fluorescent protein by adenoviral gene transfer into rat hearts in vivo. Ht31 expression resulted in loss of the striated staining pattern of type II PKA (RII), indicating loss of PKA from binding sites on endogenous AKAPs. In the absence of isoproterenol stimulation, Ht31-expressing hearts had decreased +dP/dtmax and -dP/dtmin but no change in left ventricular ejection fraction or stroke volume and decreased end diastolic pressure versus controls. This suggests that cardiac output is unchanged despite decreased +dP/dt and -dP/dt. There was also no difference in PKA phosphorylation of cardiac troponin I (cTnI), phospholamban, or ryanodine receptor (RyR2). Upon isoproterenol infusion, +dP/dtmax and -dP/dtmin did not differ between Ht31 hearts and controls. At higher doses of isoproterenol, left ventricular ejection fraction and stroke volume increased versus isoproterenol-stimulated controls. This occurred in the context of decreased PKA phosphorylation of cTnI, RyR2, and phospholamban versus controls. We previously showed that expression of N-terminal-cleaved cTnI (cTnI-ND) in transgenic mice improves cardiac function. Increased cTnI N-terminal truncation was also observed in Ht31-expressing hearts versus controls. Increased cTnI-ND may help compensate for reduced PKA phosphorylation as occurs in heart failure.

Celecoxib, a selective cyclooxygenase-2 inhibitor, decreases monocyte chemoattractant protein-1 expression and neointimal hyperplasia in the rabbit atherosclerotic balloon injury model.

The inflammation in response to vascular injury is becoming increasingly recognized as a potential contributor to restenosis. Cyclooxygenase-2 (COX-2) is the inducible form of cyclooxygenase and has been shown to be involved in the proinflammatory response of vascular tissue. Bilateral femoral artery lesions were induced by air desiccation in New Zealand White rabbits followed by high cholesterol diet feeding for 28 days. Balloon injury and stent implantation were performed at the preinjured vessel segments. Immunostaining showed that uninjured vessel segments stained positive only for COX-1 but not for COX-2. Injured vessel segments showed, in addition to COX-1, significant positive staining for COX-2. In the efficacy study, celecoxib (75 mg/kg/d) was administered orally beginning 3 hours before balloon injury or stent implantation on day 28 and daily for 21 days. Monocyte chemoattractant protein-1 (MCP-1) and matrix metalloproteinase-2 and -9 (MMPs) expression were quantified in arterial extracts 4 days after balloon injury by Western blot and gelatin zymography. Morphometric analysis and immunostaining for macrophages were performed 21 days after balloon injury. Celecoxib treatment significantly decreased MCP-1 expression (P < 0.01). Neointimal hyperplasia was significantly inhibited by celecoxib in both balloon injury and stent models (0.49 ± 0.20 versus 0.70 ± 0.35 mm2 from balloon injury model, P < 0.05, and 0.81 ± 0.25 versus 1.69 ± 0.43 mm2 from stent model, P < 0.05), accompanied by reduced macrophage infiltration. We conclude that celecoxib decreases the inflammatory response and intimal hyperplasia following vascular injury, possibly through inhibition of MCP-1 expression, implying a pivotal role of inflammation in the pathogenesis of restenosis.

Local adenoviral-mediated inducible nitric oxide synthase gene transfer inhibits neointimal formation in the porcine coronary stented model

In this study the effect of local adenoviral-mediated delivery of inducible nitric oxide synthase on restenosis was evaluated in a porcine coronary stented model. Local gene transfer of recombinant adenoviral vectors that encode human inducible nitric oxide synthase (AdiNOS) was tested. Control vector (AdNull) lacked a recombinant transgene. Endoluminal delivery of 1.0 x 10(11) adenoviral particles was accomplished in 45 s using the Infiltrator catheter (Interventional Technologies, San Diego, CA). Coronary stents were deployed, oversized by a ratio of 1.2:1, in the treated segments immediately after gene transfer. Fourteen animals were sacrificed at day 28 to evaluate the effects of iNOS gene transfer on morphometric indices, and 4 animals were sacrificed at day 4 for detection of human iNOS expression by RT-PCR. iNOS mRNA was detected in six of eight iNOS-transferred arteries, whereas no expression of human iNOS was detected in the nontarget arteries. Morphometric analysis showed that iNOS transfer significantly reduced neointimal formation (3.41 +/- 1.12 mm(2) vs 2.14 +/- 0.68 mm(2), P < 0.05). We concluded that efficient intramural adenovirus-mediated iNOS transfer can be achieved by using Infiltrator catheters. iNOS gene transfer significantly reduces neointimal hyperplasia following stent injury.

Effect of anti-tumor necrosis factor-α polyclonal antibody on restenosis after balloon angioplasty in a rabbit atherosclerotic model

Inflammation has been postulated to contribute to restenosis after balloon angioplasty. Tumor necrosis factor (TNF)-alpha is a pleiotropic proinflammatory cytokine involved in many features of inflammation. We examined the tissue expression pattern of TNF-alpha and the inflammatory response to arterial injury, and the effects of a goat anti-rabbit-TNF-alpha polyclonal antibody on tissue TNF-alpha expression, inflammation and restenosis in a rabbit atherosclerotic model. At different time points following air dessication and subsequent balloon injury, fresh rabbit femoral artery tissues were homogenized and analyzed for TNF-alpha levels by quantitative TNF-alpha bioassay. Rabbits were treated with a goat anti-rabbit-TNF-alpha polyclonal antibody, Serum and tissue TNF-alpha neutralization, macrophage infiltration (as an indicator of inflammation), and neointimal areas were determined. Balloon angioplasty increased tissue TNF-alpha expression 100000-fold over baseline, and this increase persisted over 6 days after arterial injury, serum anti-TNF-alpha antibody levels were sufficient to neutralize tissue TNF-alpha activity by 60-75%, macrophage infiltration was suppressed, but did not decrease the neointimal formation. These data indicate that tissue TNF-alpha levels were markedly increased after balloon angioplasty. Anti-TNF-alpha treatment was sufficient to neutralize tissue TNF-alpha activity, reduce inflammation, but did not inhibit neointimal formation following balloon angioplasty in a rabbit atherosclerotic model.

Administration of Recombinant P-Selectin Glycoprotein Ligand Fc Fusion Protein Suppresses Inflammation and Neointimal Formation in Zucker Diabetic Rat Model

Objective—P-selectin–mediated leukocyte-endothelium and leukocyte-platelet interaction has been reported after vascular injury and has been correlated with neointimal hyperplasia, but its role in neointimal formation after arterial injury in diabetes has not been described. Methods and Results—Using a Zucker diabetic rat balloon injury model, we examined the role of P-selectin in the vascular inflammatory process and neointimal formation after balloon injury. Immunohistochemistry revealed that P-selectin was intensely expressed and that CD45-positive leukocyte infiltration was significantly increased after arterial injury. A single preprocedural intravenous administration of a recombinant P-selectin–soluble glycoprotein ligand-Ig inhibited CD45-positive leukocyte accumulation and suppressed neointimal formation in the Zucker diabetic rat model. Conclusions—These results suggest that reduction of P-selectin–mediated leukocyte activation with the use of recombinant P-selectin–soluble glycoprotein ligand-Ig decreases the inflammatory response and limits neointimal formation after balloon injury in diabetes.