Hans Layman

Co-delivery of FGF-2 and G-CSF from gelatin-based hydrogels as angiogenic therapy in a murine critical limb ischemic model

Peripheral artery disease and critical limb ischemia have become prevalent health risks in the United States due to an increasing elderly population and the prevalence of obesity and diabetes mellitus. Although highly invasive endarterectomy is the most popular method for treatment, angiogenic therapies based on growth factor administration are quickly becoming a popular alternative. Enzymatic degradation of these factors in vivo may be avoided by their incorporation in a delivery vehicle where the growth factors release rate can be controlled by altering the vehicles properties (i.e. cross-linking density, material selection, biodegradation, etc.). Herein, we report on the immobilization and controlled release of human recombinant basic fibroblast growth factor (FGF-2) and human recombinant granulocyte colony-stimulating factor (G-CSF) from ionic, gelatin-based hydrogel scaffolds to re-establish perfusion and induce capillary outgrowth in a murine hindlimb ischemic model. In vitro studies showed that endothelial cell proliferation was highly depended on FGF-2, whereas G-CSF stimulated migration and formation of a tubular network. When FGF-2 and G-CSF were used in combination there was an 82% increase in endothelial branch point formation compared to control groups. Leg reperfusion was assessed with laser Doppler perfusion imaging, while capillary outgrowth in the ischemic leg was evaluated using CD31(+) and alpha-SMA immunostaining. The co-delivery of G-CSF (1000 ngml(-1)) and FGF-2 (1000 ng ml(-1)) from the gelatin hydrogels resulted in a 3-fold increase in the perfusion levels and a 2-fold increase in capillary density and positive alpha-SMA vessels compared to the empty vehicle group. In conclusion, the co-delivery of FGF-2 and G-CSF was superior to bolus administration or the delivery of either factor alone in promoting reperfusion and mature vessel formation.

Enhanced angiogenic efficacy through controlled and sustained delivery of FGF-2 and G-CSF from fibrin hydrogels containing ionic-albumin microspheres.

Neo-vessel formation in ischemic tissues relies on numerous growth factors and cell fractions for the formation of mature, stable, functional vasculature. However, the efforts to regenerate tissues typically rely on the administration of a single growth factor or cells alone. Conversely, polymeric matrices have been investigated extensively to deliver multiple growth factors at pre-determined rates to form stable blood vessels in ischemic tissues. We report on a novel sequential delivery system of a fibrin hydrogel containing ionic-albumin microspheres that allows for the controlled release of two growth factors. The use of this system was investigated in the context of therapeutic angiogenesis. Material properties were determined based on degree of swelling measurements and degradation characteristics. Release kinetics of model angiogenic polypeptides FGF-2 and G-CSF were determined using ELISA and the bioactivity of released protein was evaluated in human endothelial cell cultures. The release of growth factors from ionic-albumin microspheres was significantly delayed compared to the growth factor released from fibrin matrices in the absence of spheres. The scaffolds were implanted in a murine critical limb ischemia model at two concentrations, 40 ng (low) and 400 ng (high), restoring 92% of the blood flow in a normally perfused limb using a fibrin hydrogel releasing FGF-2 containing albumin–PLL microspheres releasing G-CSF (measured by LDPI at the high concentration), a 3.2-fold increase compared to untreated limbs. The extent of neo-vessel formation was delineated by immunohistochemical staining for capillary density (CD-31+) and mature vessel formation (α-SMA+). In conclusion, our study demonstrated that the release kinetics from our scaffold have distinct kinetics previously unpublished and the delivery of these factors resulted in hindlimb reperfusion, and robust capillary and mature vessel formation after 8 weeks compared to either growth factor alone or bolus administration of growth factor.

Vascular Regeneration in Ischemic Hindlimb by Adeno‐Associated Virus Expressing Conditionally Silenced Vascular Endothelial Growth Factor

Background Critical limb ischemia (CLI) is the extreme manifestation of peripheral artery disease, a major unmet clinical need for which lower limb amputation is the only option for many patients. After 2 decades in development, therapeutic angiogenesis has been tested clinically via intramuscular delivery of proangiogenic proteins, genes, and stem cells. Efficacy has been modest to absent, and the largest phase 3 trial of gene therapy for CLI reported a worsening trend of plasmid fibroblast growth factor. In all clinical trials to date, gene therapy has used unregulated vectors with limited duration of expression. Only unregulated extended expression vectors such as adeno‐associated virus (AAV) and lentivirus have been tested in preclinical models. Methods and Results We present preclinical results of ischemia (hypoxia)‐regulated conditionally silenced (CS) AAV–human vascular endothelial growth factor (hVEGF) gene delivery that shows efficacy and safety in a setting where other strategies fail. In a BALB/c mouse model of CLI, we show that gene therapy with AAV‐CS‐hVEGF, but not unregulated AAV or plasmid, vectors conferred limb salvage, protection from necrosis, and vascular regeneration when delivered via intramuscular or intra‐arterial routes. All vector treatments conferred increased capillary density, but organized longitudinal arteries were selectively generated by AAV‐CS‐hVEGF. AAV‐CS‐hVEGF therapy reversibly activated angiogenic and vasculogenic genes, including Notch,SDF1, Angiopoietin, and Ephrin‐B2. Reoxygenation extinguished VEGF expression and inactivated the program with no apparent adverse side effects. Conclusions Restriction of angiogenic growth factor expression to regions of ischemia supports the safe and stable reperfusion of hindlimbs in a clinically relevant murine model of CLI.

Sustained Endothelial Expression of HoxA5 In Vivo Impairs Pathological Angiogenesis And Tumor Progression

HoxA5 is expressed in quiescent endothelial cells (EC), but absent in activated angiogenic EC. To examine the efficacy of targeting HoxA5 therapeutically to quell pathologic or tumor angiogenesis, we generated an inducible, transgenic mouse model of sustained HoxA5 expression in ECs. During pathologic angiogenesis, sustained HoxA5 regulates expression several angiogenic effector molecules, notably increased expression of TSP-2 and reduced expression of VEGF, thus leading to inhibition of pathological angiogenesis in tissues. To evaluate if this impressive reduction of vascularization could also impact tumor angiogenesis, HoxA5 mice were bred with a mouse model of de novo squamous carcinogenesis, e.g., K14-HPV16 mice. Activation of EC-HoxA5 significantly reduced infiltration by mast cells into neoplastic skin, an early hallmark of progression to dysplasia, reduced angiogenic vasculature, and blunted characteristics of tumor progression. To evaluate HoxA5 as a therapeutic, topical application of a HoxA5 transgene onto early neoplastic skin of K14-HPV16 mice similarly resulted in a significant impairment of angiogenic vasculature and progression to dysplasia to a similar extent as observed with genetic delivery of HoxA5. Together these data indicate that HoxA5 represents a novel molecule for restricting pathological and tumorigenic angiogenesis.

Whole-mount imaging of the mouse hindlimb vasculature using the lipophilic carbocyanine dye DiI.

The availability of transgenic disease backgrounds and the accessibility of molecular research reagents have contributed to make the mouse ischemic hindlimb the model of choice for many studies of angiogenesis, and to investigate new treatments for peripheral artery disease. A limitation of these models involves our inability to easily visualize the regenerated vascular architecture. Approaches such as micro-computed tomography and micro-angiography are expensive, technically demanding and not available to many laboratories. Here we describe a rapid and inexpensive adaptation of a vascular staining procedure for precise imaging of the mouse hindlimb vasculature. We introduced two technical modifications and an analytical extension to the original method including (i) pre-skinning of the muscle prior to fixation that preserves tissue integrity, (ii) mild pressure-desiccation subsequent to fixing that enhances resolution and image penetration, and (iii) reconstruction of confocal data into 3D images. The procedure provides resolution that is equivalent or superior to other approaches at a fraction of the cost, time and technology required.

Abstract A07: Breast tumor microenvironment shapes vascular response to endothelial HoxA5 expression

Current anti-angiogenic therapy for the treatment of solid tumors is based on directed inhibition of growth factor signaling pathways essential for the development of new blood vessels. Despite evidence showing a positive correlation between angiogenesis and breast cancer progression, existing anti-angiogenic therapies have not proven effective at clinically managing breast tumors or prolonging patient survival. Several studies have shown that tissue microenvironment shapes local angiogenic responses and tumor progression - a finding that may partially explain the refractoriness of breast tumors to anti-angiogenic therapy. Data from our laboratory supports an anti-angiogenic role for the HoxA5 homeodomain containing transcription factor. Using the KRT14-HPV16 mouse model of skin cancer crossed with our tetracycline-regulated mouse line that expresses HoxA5 in the endothelium, we observed that HoxA5 reduced angiogenesis and slowed tumor progression in the skin. Thus, we hypothesized that constitutive endothelial expression of HoxA5 during mammary tumor development would prevent tumor growth and metastasis via modulation of the endothelial phenotype. Surprisingly, in the MMTV-PyMT mouse model of breast cancer, we observed that endothelial HoxA5 expression increased both primary tumor growth and lung metastasis. Mammary tumors from PyMT/HoxA5+ mice had larger areas of hypoxia and necrosis, as compared to primary tumors from control mice. Although we did not detect any change in intra-tumoral vascular staining (CD31+), we observed an increased number of large vessels and reduced vascular leakage in the primary tumors from PyMT/HoxA5+ mice. In contrast, subcutaneous injection of MMTV-PyMT mammary tumor cells into HoxA5 transgenic mice displayed significantly reduced tumor growth and decreased intra-tumoral vascularization, as compared to controls. This suggests that, unlike the anti-angiogenic properties of HoxA5 in the skin microenvironment, within the context of the mammary gland HoxA5 has vascular normalization effects. We conclude that the tissue microenvironment shapes the vascular response to anti-angiogenic agents and thereby controls tumor progression. Citation Format: Josette Northcott, Ileana Cuevas, Hans Layman, Nancy Boudreau. Breast tumor microenvironment shapes vascular response to endothelial HoxA5 expression. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A07. doi:10.1158/1538-7445.CHTME14-A07