Caroline C. Jadlowiec

Toward A Mouse Model of Hind Limb Ischemia to Test Therapeutic Angiogenesis

INTRODUCTION Several clinical trials are currently evaluating stem cell therapy for patients with critical limb ischemia that have no other surgical or endovascular options for revascularization. However, these trials are conducted with different protocols, including use of different stem cell populations and different injection protocols, providing little means to compare trials and guide therapy. Accordingly, we developed a murine model of severe ischemia to allow methodic testing of relevant clinical parameters. METHODS High femoral artery ligation and total excision of the superficial femoral artery was performed on C57BL/6 mice. Mononuclear cells (MNCs) were isolated from the bone marrow of donor mice, characterized using fluorescence-activated cell sorting, and injected (5×10(5) to 2×10(6)) into the semimembranosus (proximal) or gastrocnemius (distal) muscle. Vascular and functional outcomes were measured using invasive Doppler imaging, laser Doppler perfusion imaging, and the Tarlov and ischemia scores. Histologic analysis included quantification of muscle fiber area and number as well as capillary density. RESULTS Blood flow and functional outcomes were improved in MNC-treated mice compared with controls over 28 days (flow: P<.0001; Tarlov: P=.0004; ischemia score: P=.0002). MNC-treated mice also showed greater gastrocnemius fiber area (P=.0053) and increased capillary density (P=.0004). Dose-response analysis showed increased angiogenesis and gastrocnemius fiber area but no changes in macroscopic vascular flow or functional scores. Overall functional outcomes in mice injected proximally to the ischemic area were similar to mice injected more distally, but muscle flow, capillary density, and gastrocnemius fiber area were increased (P<.05). CONCLUSIONS High femoral ligation with complete excision of the superficial femoral artery is a reliable model of severe hind limb ischemia in C57BL/6 mice that shows a response to MNC treatment for functional and vascular outcomes. A dose response to the injection of MNCs appears to be present, at least microscopically, suggesting that an optimal cell number for stem cell therapy exists and that preclinical testing needs to be performed to optimally guide human trials. Injection of MNCs proximal to the site of ischemia may provide different outcomes compared with distal injection and warrants additional study.

Therapeutic strategies to combat neointimal hyperplasia in vascular grafts

Neointimal hyperplasia (NIH) in bypass conduits such as veins and prosthetic grafts is an important clinical entity that limits the long-term success of vascular interventions. Although the development of NIH in the conduits shares many of the same features of NIH that develops in native arteries after injury, vascular grafts are exposed to unique circumstances that predispose them to NIH, including surgical trauma related to vein handling, hemodynamic changes creating areas of low flow, and differences in biocompatibility between the conduit and the host environment. Multiple different approaches, including novel surgical techniques and targeted gene therapies, have been developed to target and prevent the causes of NIH. Recently, the PREVENT trials, the first molecular biology trials in vascular surgery aimed at preventing NIH, have failed to produce improved clinical outcomes, highlighting the incomplete knowledge of the pathways leading to NIH in vascular grafts. In this review, we aim to summarize the pathophysiologic pathways that underlie the formation of NIH in both vein and synthetic grafts and discuss current and potential mechanical and molecular approaches under investigation that may limit NIH in vascular grafts.

Stem cell therapy for critical limb ischemia: What can we learn from cell therapy for chronic wounds?

Although much progress has been made regarding our knowledge of stem cells and their potential applications for therapeutic angiogenesis, there has been less success with the clinical application of this knowledge to patients with critical limb ischemia (CLI). Patients with CLI often have chronic wounds and newer cell-based therapies for chronic wounds show interesting parallels to stem cell therapy for CLI. Several human-derived wound care products and therapies, including human neonatal fibroblast-derived dermis (Dermagraft®), bilayered bioengineered skin substitute (Apligraf®), recombinant human platelet-derived growth factor and autologous platelet-rich plasma may provide insight into the mechanisms through which differentiated cells can be used as therapy for chronic wounds, and, analogously, by which stem cells might function therapeutically in CLI.

Cell-based interventions for therapeutic angiogenesis: review of potential cell sources

Alternative therapies are currently being developed to treat patients with chronic limb ischemia who are unable to be revascularized in order to avoid amputation. Cell-based therapy using mononuclear cells is gaining attention as many clinical trials are currently underway. We review cell differentiation along with the different potential cell sources for use in therapeutic angiogenesis.

Vascular endothelial growth factor-A inhibits EphB4 and stimulates delta-like ligand 4 expression in adult endothelial cells

BACKGROUND During vein graft adaptation to the arterial circulation, vascular endothelial growth factor (VEGF) A expression transiently increases before becoming downregulated; however, the role of VEGF-A in venous remodeling is not clear. In addition, although VEGF-A stimulates angiogenesis and determines arterial identity in nascent arterial endothelial cells (EC), the role of VEGF-A in regulating identity in adult venous EC is also not clear. MATERIALS AND METHODS EC, wild type (EphB4+/+) or heterozygous knockout (EphB4+/-), were stimulated with VEGF-A (0-100 ng/mL) and examined with quantitative polymerase chain reaction and western blotting. RESULTS VEGF-A (100 ng/mL) inhibited expression of EphB4 and stimulated expression of delta-like ligand 4 (dll4) but did not stimulate either notch or EphrinB2 expression in adult venous EC. Pretreatment with VEGF receptor 2-neutralizing antibody abolished VEGF-stimulated downregulation of EphB4 but not the upregulation of dll4. Pretreatment with PD98059 or wortmannin showed that VEGF-A downregulation of EphB4 and upregulation of dll4 are mitogen-activated protein kinase kinase and extracellular signal-regulated kinase dependent but phosphatidylinositol 3 kinase-Akt independent. Compared with VEGF-induced EphB4 downregulation and dll4 upregulation in control EC, reduced EphB4 signaling in EphB4+/- EC showed even further downregulation of EphB4 and upregulation of dll4. CONCLUSIONS Despite the genetic programming of arterial and venous EC fate, VEGF-A can repress venous identity in adult venous EC without induction of arterial identity. These changes in adult EC in vitro recapitulate the changes in identity described during vein graft adaptation to the arterial environment in vivo.

Pericardial Patch Angioplasty Heals via an Ephrin-B2 and CD34 Positive Cell Mediated Mechanism

Objective Pericardial patches are commonly used in vascular surgery to close arteriotomies. The mechanism of early healing after patch implantation is still not well defined. We used a rat aortic patch model to assess pericardial patch healing and examined Ephrin-B2, a marker of arterial identity, expression within the post-implantation patch. We also determined whether endothelial progenitor cells (EPC) are associated with early patch healing in the arterial environment. Methods Wistar rats (200–250 grams) underwent infrarenal aortic arteriotomy and then closure via bovine or porcine pericardial patch angioplasty. Control groups included subcutaneously implanted patches. Patches were harvested at 0–30 days and analyzed by histology, immunohistochemistry, immunofluorescence and Western blot as well as quantitative PCR. Results Prior to implantation, pericardial patches are largely composed of collagen and are acellular. Following arterial implantation, increasing numbers of CD68-positive cells as well as Ephrin-B2 and CD34 dual-positive cells are found within both bovine and porcine pericardial patches, whereas the infiltrating cells are negative for vWF and α-actin. Porcine patches have a luminal monolayer of cells at day 7, compared to bovine patches that have fewer luminal cells. Subcutaneously implanted patches do not attract Ephrin-B2/CD34-positive cells. By day 30, both bovine and porcine pericardial patches develop a neointima that contains Ephrin-B2, CD34, and VEGFR2-positive cells. Conclusion Both CD68-positive and Ephrin-B2 and CD34 dual-positive cells infiltrate the pericardial patch early after implantation. Arteriotomy closure via pericardial patch angioplasty shows patch adaptation to the arterial environment that may involve a foreign body response as well as localization of EPC. Arterial remodeling of pericardial patches support endothelialization and may represent a paradigm of healing of scaffolds used for tissue engineering.

The Nogo-B-PirB axis controls macrophage-mediated vascular remodeling.

Objective Nogo-B mediates vascular protection and facilitates monocyte- and macrophage-dependent vascular remodeling. PirB is an alternate receptor for Nogo-B, but a role for the Nogo-PirB axis within the vascular system has not been previously reported. We examined whether Nogo-B or PirB play a role in regulating macrophage-mediated vascular remodeling and hypothesized that endothelial Nogo-B regulates vein graft macrophage infiltration via its alternate receptor PirB. Methods Vein grafts were performed using Nogo and PirB wild type and knockout mice. Human vein grafts were similarly analyzed. The hindlimb ischemia model was performed in PirB wild type and knockout mice. Accompanying in vitro work included isolation of macrophages from PirB wild type and knockout mice. Results Increased Nogo-B and PirB mRNA transcripts and protein expression were observed within mouse and human vein grafts. Both Nogo knockout and PirB knockout vein grafts showed increased wall thickness and increased numbers of F4/80-positive macrophages. Macrophages derived from PirB knockout mice had increased adhesion to fibronectin, increased EC-specific binding, and increased numbers of mRNA transcripts of M2 markers as well as MMP3 and MMP9. PirB knockout vein grafts had increased active MMP9 compared to wild type vein grafts. PirB knockout mice had increased recovery from hindlimb ischemia and increased macrophage infiltration compared to wild type mice. Conclusions Vein graft adaptation shows increased expression of both Nogo-B and PirB. Loss of PirB, or its endothelial ligand Nogo-B, results in increased inflammatory cell infiltration and vein graft wall thickening. These findings suggest that PirB regulates macrophage activity in vein grafts and that Nogo-B in the vein graft limits macrophage infiltration and vein graft thickening. PirB may play a more general role in regulating macrophage responses to vascular injury. Macrophage inhibition via Nogo-PirB interactions may be an important mechanism regulating vein graft adaptation to the arterial circulation.

Reduced adult endothelial cell EphB4 function promotes venous remodeling

Reduced EphB4 expression is observed during vein graft adaptation and is associated with increased venous wall thickening. These findings suggest that EphB4 may mediate normal adult venous endothelial cell (EC) function and vein graft adaptation. We therefore tested the functional significance of EphB4 using EC with genetically reduced EphB4 signaling. EC were isolated from EphB4(+/+) and EphB4(+/-) mice. In vitro function was assessed through EC proliferation, migration, nitric oxide (NO) synthesis, and chemokine production. A mouse vein graft model was used to correlate in vitro findings with in vivo vein grafts. Smooth muscle cells (SMC) were subjected to proliferation and migration assays using EphB4(+/+) and EphB4(+/-) EC-conditioned medium. EphB4(+/-) EC exhibited diminished proliferation (P < 0.0001, n = 6), migration (P < 0.0001, n = 3), and NO production (P = 0.0012, n = 3). EphB4(+/-) EC had increased VEGF-A mRNA (P = 0.0006, n = 6) and protein (P = 0.0106, n = 3) as well as increased secretion of VEGF-A (P = 0.0010, n = 5), PDGF-BB (P < 0.0001, n = 6), and TGF-β1 (P < 0.0001, n = 6). EphB4(+/-)-conditioned medium promoted SMC proliferation (P < 0.0001, n = 7) and migration (P = 0.0358, n = 3). Vein grafts and EphB4(+/-) EC showed similarity with regard to VEGF-A and eNOS mRNA and protein expression. In conclusion, reduced venous EC EphB4 function is associated with a proangiogenic and mitogenic phenotype. EphB4(+/-) EC have increased secretion of SMC mitogens and reduced NO production that correlate with the thickened neointima formed during vein graft adaptation. These findings suggest that EphB4 remains active in adult venous EC and that loss of EphB4 plays a role in vein graft adaptation.

Shear Stress and Endothelial Cell Retention in Critical Lower Limb Ischemia

Progressive atherosclerotic stenosis of vessels commonly leads to the development of critical limb and myocardial ischemia. When possible and appropriate, surgical revascularization is attempted, and it is here that we clinically observe the pathological processes of ischemia and reperfusion and their complex effects [1]. Understanding of the role and function of the vascular endothelium has undergone significant changes over the past several decades. In the 1960s Willms-Kretschmer and colleagues referred to altered endothelial cells as being activated and, in doing so, implied a functional consequence to the altered cell morphology [2, 3]. This dynamic view of the endothelium, however, did not ensue into the following decade when, again, it was believed that endothelial cells were nothing more than a passive barrier. It would not be until the 1980s that Pober and colleagues would reexamine the scientific principle and ultimately prove that the vascular endothelium is both dynamic and integral to vascular and systemic equilibrium [4]. The scientific process to better understand the endothelium dates back to the 1800s when von Recklinghausen recognized that vessels were not merely inert tunnels passing through tissue, but living entities lined by cells [5]. The endothelial monolayer comprises the entirety of the vascular system, and it is now recognized that the diversity of these cells is not merely limited by cell type alone, but rather is a function of anatomic hemodynamic variation. The unique interface formed by the endothelium between blood and the surrounding vessel wall allows it to function as a primary mediator in response to shear stress alterations.