Pro-angiogenic Compounds Research Articles

Delivery of Bioactive Compounds to Improve Skin Cell Responses on Microfabricated Electrospun Microenvironments.

The introduction of microtopographies within biomaterial devices is a promising approach that allows one to replicate to a degree the complex native environment in which human cells reside. Previously, our group showed that by combining electrospun fibers and additive manufacturing it is possible to replicate to an extent the stem cell microenvironment (rete ridges) located between the epidermal and dermal layers. Our group has also explored the use of novel proangiogenic compounds to improve the vascularization of skin constructs. Here, we combine our previous approaches to fabricate innovative polycaprolactone fibrous microtopographical scaffolds loaded with bioactive compounds (2-deoxy-D-ribose, 17β-estradiol, and aloe vera). Metabolic activity assay showed that microstructured scaffolds can be used to deliver bioactive agents and that the chemical relation between the working compound and the electrospinning solution is critical to replicate as much as possible the targeted morphologies. We also reported that human skin cell lines have a dose-dependent response to the bioactive compounds and that their inclusion has the potential to improve cell activity, induce blood vessel formation and alter the expression of relevant epithelial markers (collagen IV and integrin β1). In summary, we have developed fibrous matrixes containing synthetic rete-ridge-like structures that can deliver key bioactive compounds that can enhance skin regeneration and ultimately aid in the development of a complex wound healing device.

Development of a dual screening strategy to identify pro-angiogenic compounds from natural products: application on Tongmai Yangxin Pills

Establishment and application the dual-screening strategy to screen pro-angiogenic compounds from natural products for the first time.

Pro-Angiogenic Effects of Chalcone Derivatives in Zebrafish Embryos in Vivo.

The aim of this study was to investigate novel chalcones with potent angiogenic activities in vivo. Chalcone-based derivatives were evaluated using a transgenic zebrafish line with fluorescent vessels to real-time monitor the effect on angiogenesis. Results showed that the chalcone analogues did not possess anti-angiogenic effect on zebrafish vasculatures; instead, some of them displayed potent pro-angiogenic effects on the formation of the sub-intestinal vein. Similar pro-angiogenic effects can also be seen on wild type zebrafish embryos. Moreover, the expression of vegfa, the major regulator for angiogenesis, was also upregulated in their treatment. Taken together, we have synthesized and identified a series of novel chalcone-based derivatives as potent in vivo pro-angiogenic compounds. These novel compounds hold potential for therapeutic angiogenesis.

Pro-angiogenic and Angiostatic Compounds from Terrestrial and Marine Sources

The formation of new blood vessels out of pre-existing capillaries or angiogenesis is a sequence of events that is of key importance in a broad array of physiologic and pathologic processes. In several diseases, excessive angiogenesis is a part of the pathology such as cancer (both solid and hematologic tumors), cardiovascular disease (atherosclerosis), chronic inflammation (rheumatoid arthritis, Crohn’s disease), diabetes (proliferative diabetic retinopathy), neovascular wet age related macular degeneration, retinopathy of prematurity, psoriasis, andAIDS complications. These diseases may benefit from the therapeutic inhibition of angiogenesis. There has been a substantial progress made in research on natural products possessing several therapeutic actions as a result of which they have attracted attention of scientists, the world over and has profoundly influenced the course of discovery from traditional remedies. The present review entails such natural products from terrestrial and marine sources that have exhibited pro-angiogenic or antiangiogenic effects and offer subsequent potential for the development of novel pharmacological agents in the near future.

ABT-898 Induces Tumor Regression and Prolongs Survival in a Mouse Model of Epithelial Ovarian Cancer

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy and is often not diagnosed until late stages due to its asymptomatic nature. Women diagnosed with EOC typically undergo surgical debulking followed by chemotherapy; however, disease recurrence often occurs. In this study, we evaluated the ability of the thrombospondin-1 mimetic peptide, ABT-898, to regress established, late-stage tumors in a mouse model of human EOC. Ovarian tumors were induced and ABT-898 treatment was initiated at time points that were representative of late stages of the disease to study tumor regression. ABT-898 induced tumor regression and reduced the morbidity of treated animals compared with controls. Analysis of tumors from ABT-898-treated animals showed reduced abnormal tumor vasculature, decreased expression of the proangiogenic compound VEGF, and reduced tumor tissue hypoxia. ABT-898 treatment initiated at late-stage disease also significantly prolonged disease-free survival compared with control animals. Results from this study show that ABT-898 is capable of regressing established ovarian tumors in an animal model of the disease. As most women are detected at advanced stage EOC, ABT-898 may improve our treatment of ovarian cancer.

121 Identification, validation and mechanisms of action of novel therapeutic compounds modulating angiogenesis

Abnormal angiogenesis is a signature feature in over 70 health conditions and is critical for tumour growth and metastasis. Therefore, discovering novel therapeutic compounds, that stimulate or abrogate this biological process would be, potentially, clinically invaluable. The zebrafish has emerged as an important model for studying vascular development and angiogenesis. Advantages over other models include rapid embryonic development and optical clarity of the embryos. The small body size of the zebrafish embryo and accessibility of embryonic developmental structures within water make zebrafish embryos suitable for high through-put drug screens. We performed an angiogenesis chemical genetic screen with two compound libraries (LOPAC and Spectrum Collection) using Flk1:EGFP (fluorescently labelled endothelial cells) transgenic zebrafish embryos. The compound bioactivity was measured by numbers of the zebrafish trunk intersegmental vessel (ISV) number. The screens were performed in 96 well plates and a total of ten antiangiogenic compounds and three proangiogenic compounds were identified. A CFI-funded high throughput screening facility will be use for identification of more lead compounds. The goal of the current study was to validate the potential therapeutic efficacy of selected compounds and investigate their molecular mechanisms. Retinoic acid p-hydroxyanilide (4HPR; Fenretinide) was identified as a pro-angiogenic compound. At 4 μM, 4HPR promoted ISV development with increased ISV numbers (ISV = 32.82 ± 0.98; control ISV = 27.58 ± 0.76; P < 0.05). In HUVEC cell proliferation assay, 4HPR stimulated human endothelial cell proliferation at low molar concentrations (0.06-0.12 μM). The effects of this compound on physiological angiogenesis are currently being tested on larvae and adult zebrafish fin regeneration models. Indirubin-3-oxime (IO) was identified as a very potent antiangiogenic compound, which inhibited zebrafish ISV development in a dose-dependent manner (Fig. 1). At 16 μM, IO almost completely inhibited ISV development. In tissue regeneration models, IO inhibited larval and adult zebrafish fin regeneration. The efficacy of IO in cancer therapy will be further studied in a zebrafish cancer metastasis model. Possible molecular mechanisms responsible for IO and 4HPR action such as VEGF and notch signaling will be studied. Using the zebrafish chemical genetic screen, we demonstrated that indirubin-3-oxime is a potent angiogenic inhibitor which inhibits zebrafish angiogenesis and fin regeneration. IO may prove to be effective as an anti-cancer drug. Retinoic acid p-hydroxyanilide was identified as a pro-angiogenic effector, which may be clinically effective in promoting tissue regeneration and in treatment of myocardial infarction.

Necrotic cell death in atherosclerosis

Necrosis is a type of cell death characterized by a gain in cell volume, swelling of organelles, rupture of the plasma membrane and subsequent loss of intracellular contents. For a long time, the process has been considered as a merely accidental and uncontrolled form of cell death, but accumulating evidence suggests that it can also occur in a regulated fashion. Morphological studies using transmission electron microscopy indicate that the vast majority of dying cells in advanced human atherosclerotic plaques undergo necrosis. Various stimuli in the plaque including high levels of oxidative stress, depletion of cellular ATP, impaired clearance of apoptotic cells and increased intracellular calcium may cause necrotic death. Although the role of necrosis in atherosclerosis remains ill-defined, a growing body of evidence suggests that necrotic death stimulates atherogenesis through induction of inflammation and enlargement of the necrotic core. In addition, necrosis contributes to plaque instability by releasing tissue factor, matrix degrading proteases and pro-angiogenic compounds. Therapeutic agents against necrosis are limited, but efforts have recently been made to inhibit the necrotic pathway or its pro-inflammatory effects.

PLGA:poloxamer blend micro- and nanoparticles as controlled release systems for synthetic proangiogenic factors

Tissue engineering is one of the most promising research areas in bioregenerative medicine. However, the restoration of biological functionalities by implanting bioartificially engineered tissues is still highly limited because of their lack of vascular networks. The use of proangiogenic molecules delivered from a controlled release device is a promising strategy to induce tissue vascularization. Indeed, the controlled release system can enhance the therapeutic effect in vivo of many short half-life drugs, while circumventing the need for repeated administrations. In this work, PLGA:poloxamer blend based micro- and nanoparticles have been developed for the sustained delivery of a recently developed synthetic proangiogenic compound: SHA-2-22. Drug-loaded PLGA:poloxamer blend microparticles were prepared by an oil-in-oil solvent extraction/evaporation technique. Drug-loaded PLGA:poloxamer nanoparticles were prepared by a modified solvent diffusion technique. These drug carriers were characterized with regard to their physicochemical properties, morphology, drug encapsulation efficiency and release kinetics in vitro. The results show that by adjusting the formulation conditions, it is possible to obtain PLGA:poloxamer micro- and nanoparticles with very high drug loadings, and with the capacity to release the active compound in a controlled way for up to one month. In vitro cell assays performed in an endothelial cell model confirmed the bioactivity of SHA-22-2 encapsulated in PLGA:poloxamer microparticles.

Targeting VEGF‐encapsulated immunoliposomes to MI heart improves vascularity and cardiac function

Recent attempts at rebuilding the myocardium using stem cells have yielded disappointing results. The lack of a supporting vasculature may, in part, explain these disappointing findings. However, concerns over possible side effects have hampered attempts at revascularizing the infarcted myocardium using systemic delivery of proangiogenic compounds. In this study, we develop the technology to enhance the morphology and function of postinfarct neovasculature. Previously, we have shown that the up-regulated expression of endothelial cell adhesion molecules in the myocardial infarction (MI) region provides a potential avenue for selectively targeting drugs to infarcted tissue. After treatment with anti-P-selectin-conjugated liposomes containing vascular endothelial growth factor (VEGF), changes in cardiac function and vasculature post-MI were quantified in a rat MI model. Targeted delivery of VEGF to post-MI tissue resulted in significant increase in fractional shortening and improved systolic function. These functional improvements were accompanied by a 21% increase in the number of anatomical vessels and a 74% increase in the number of perfused vessels in the MI region of treated animals. No significant improvements in cardiac function were observed in untreated, systemic VEGF-treated, nontargeted liposome-treated, or blank immunoliposome-treated animals. Targeted delivery of low doses of proangiogenic compounds to post-MI tissue results in significant improvements in cardiac function and vascular structure.

Acceleration of Diabetic Wound Healing by an Angiopoietin Peptide Mimetic

Angiopathies are one of the leading underlying causes of morbidity in diabetic patients. Poorly managed blood glucose levels contribute to vascular defects that manifest themselves in numerous different clinical conditions, including diabetic retinopathy, nephropathy, peripheral artery disease, and compromised wound healing. The angiopoietin family (Angs 1-4) has been shown to play a critical role in the growth and maintenance of vasculature. Here we evaluate the efficacy of a new Ang-based peptidomimetic compound, Vasculotide, on diabetic-related wound healing. Stimulation of endothelial cells (ECs) with Vasculotide results in activation of the Ang receptor, Tie 2, and its associated signaling pathways. This activation promoted biological responses such as EC survival, migration, and matrix metalloproteinase 2 (MMP2) production. We show that Vasculotide alone and in combination with vascular endothelial growth factor (VEGF) results in the production of well-arborized vessels supported by myogenic cells. Using an excisional skin-wound model produced on the back of diabetic B6.Cg-m(+/+)Lepr(db)/J (db/db) mice, we found that Vasculotide-treated wounds presented with decreased wound closure times (p < 0.05) and dramatic increases in granulation tissue (p < 0.01). Although the potential of this novel proangiogenic compound in treating microvascular dysfunction is not strictly limited to topical administration, we provide mechanistic evidence as a proof of principle in support of its efficacious use in diabetic wound healing.

Vascular Regeneration by Local Growth Factor Release Is Self-Limited by Microvascular Clearance

The challenge of angiogenesis science is that stable sustained vascular regeneration in humans has not been realized despite promising preclinical findings. We hypothesized that angiogenic therapies powerfully self-regulate by dynamically altering tissue characteristics. Induced neocapillaries increase drug clearance and limit tissue retention and subsequent angiogenesis even in the face of sustained delivery. We quantified how capillary flow clears fibroblast growth factor after local epicardial delivery. Fibroblast growth factor spatial loading was significantly reduced with intact coronary perfusion. Penetration and retention decreased with transendothelial permeability, a trend diametrically opposite to intravascular delivery, in which factor delivery depends on vascular leak, but consistent with a continuum model of drug transport in perfused tissues. Model predictions of fibroblast growth factor sensitivity to manipulations of its diffusivity and transendothelial permeability were validated by conjugation to sucrose octasulfate. Induction of neocapillaries adds pharmacokinetic complexity. Sustained local fibroblast growth factor delivery in vivo produced a burst of neovascularization in ischemic myocardium but was followed by drug washout and a 5-fold decrease in fibroblast growth factor penetration depth. The very efficacy of proangiogenic compounds enhances their clearance and abrogates their pharmacological benefit. This self-limiting property of angiogenesis may explain the failures of promising proangiogenic therapies.

Use of a Nanoflex powder dressing for wound management following debridement for necrotising fasciitis in the diabetic foot

This paper discusses the application of Nanoflex powder dressing for management of complex soft tissue wounds. A case report is presented detailing the management of a 43-year-old Native American woman with diabetes mellitus who required serial debridements for necrotising fasciitis. Following debridement, the patient was left with a large dorsal foot wound and was transitioned through multiple advanced wound healing modalities. Negative pressure wound therapy (NPWT) was initially utilised in the early postoperative setting to control drainage and to promote granulation tissue; the patient was subsequently transitioned to a Nanoflex powder dressing on postoperative day 4. She reported a decrease in pain associated with dressing changes when transitioned from NPWT to the use of Nanoflex powder dressing. We hypothesise that this pain reduction is the result of a light cooling effect of the exudate-controlling dressing and subsequent reduction in inflammation as well as the total contact nature of the dressing. Nanoflex powder dressings are a recently developed advanced wound healing modality with promise in the management of complex soft tissue wounds, both as a primary wound dressing as well as a delivery platform for analgesics, antimicrobials and pro-angiogenic compounds.

A Chemical Genetic Screen Identified Small Molecules Modulating Zebrafish Vascular Development and Angiogenesis.

The zebrafish has emerged as an important model for studying vascular development and angiogenesis. Advantages over other models include rapid embryonic development and optical clarity of the embryos. Abnormal angiogenesis is linked to over 70 health conditions and inhibition of angiogenesis is an excellent target for cancer therapy (including hematopoietic malignancies) as tumor growth requires new blood vessels. In this study, we performed a chemical genetic screen in developing zebrafish embryos to identify compounds that modulate zebrafish vascular development and angiogenesis. A zebrafish transgenic line with the Flk1 promoter controlling the GFP (green fluorescent protein) reporter was used. The entire vascular network of the Flk1:GFP fish was marked by GFP and could be visualized under fluorescence microscopy. Screens were performed in 96 well plates with three embryos/well. The outer membranes of healthy zebrafish embryos expressing green fluorescent vasculature were removed by enzymatic digestion with protease at the 16–18 somite stages. Small molecules/compounds were then added to the water at concentrations of 0.1 μM, 1 μM, and 5 μM for 72 hrs. After 24, 48 and 72 hrs of exposure to the compound, the embryos were visually inspected for viability, gross morphological defects, heartbeating rate and circulation. The known angiogenic inhibitor, PD173074 was used as control. An initial screening of 780 compounds in a small molecule compound library identified four small molecules with potent activities in inhibiting zebrafish vascular development. Notably, 1–5 μM of Hit #1 inhibited the growth of cranial vessels and disrupted vascular patterning, which resulted in uneven spacing between intersegmental vessels. While Hit #2 and #5 also inhibited the growth of cranial vessels, the vascular patterning remained unaffected, however, the drug-treated embryos had weak or missing intersegmental vessels. Fish embryos treated by Hit #3 had an enlarged heart and thinner vessels. Interestingly, this screen also identified one compound (Hit #4) with pro-angiogenic activity. Embryos treated with Hit #4 had increased numbers of intersegmental vessels. Hit #2 is a known inhibitor of c-Jun N-terminal kinase and angiogenesis (SP600125), was recently reported to inhibit the proliferation and migration of human endothelial cells in vitro as well as inhibit solid tumor growth in mice. This observation lends validity to the zebrafish screen and the potential utility of the other hits. Experiments are currently in progress to investigate the detailed time course of the angiogenic inhibition and the potential molecular mechanism. Studies of these angiogenic inhibitors may lead to the development of potent anti-cancer drugs while the pro-angiogenic compound may prove useful in facilitating tissue/organ regeneration. We conclude that the zebrafish model is likely to yield valuable information regarding vasculogenesis and its manipulation.

Microvascular transport model predicts oxygenation changes in the infarcted heart after treatment

Chronic heart failure is most commonly due to ischemic cardiomyopathy after a previous myocardial infarction (MI). Rebuilding lost myocardium to prevent heart failure mandates a neovasculature able to nourish new cardiomyocytes. Previously we have used a series of novel techniques to directly measure the ability of the scar neovasculature to deliver and exchange oxygen at 1-4 wk after MI in rats following left coronary artery ligation. In this study, we have developed a morphologically realistic mathematical model of oxygen transport in cardiac tissue to help in deciding what angiogenic strategies should be used to rebuild the vasculature. The model utilizes microvascular morphology of cardiac tissue based on available morphometric images and is used to simulate experimentally measured oxygen levels after MI. Model simulations of relative oxygenation match experimental measurements closely and can be used to simulate distributions of oxygen concentration in normal and infarcted rat hearts. Our findings indicate that both vascular density and vascular spatial distribution play important roles in cardiac tissue oxygenation after MI. Furthermore, the model can simulate relative changes in tissue oxygen levels in infarcted tissue treated with proangiogenic compounds such as losartan. From the minimum oxygen concentration myocytes need to maintain their normal function, we estimate that 2 wk after MI 29% of the myocardium is severely hypoxic and that the vascular density of the infarcted tissue should reach 75% of normal tissue to ensure that no areas of the myocardium are critically hypoxic.

Targeted delivery of antibody conjugated liposomal drug carriers to rat myocardial infarction

Immunoliposome (IL) targeting to areas of inflammation after an acute myocardial infarction (MI) could provide the means by which pro-angiogenic compounds can be selectively targeted to the infarcted region. The adhesion of model drug carriers and ILs coated with an antibody to P-selectin was quantified in a rat model of MI following left coronary artery ligation. Anti-P-selectin coated model drug carriers showed a 140% and 180% increase in adhesion in the border zone of the MI 1 and 4 h post-MI, respectively. Radiolabeled anti-P-selectin ILs injected immediately post-MI and allowed to circulate 24 h showed an 83% increase in targeting to infarcted myocardium when compared to adjacent non-infarcted myocardium. Radiolabeled anti-P-selectin ILs injected 4 h post-MI and allowed to circulate for 24 h showed a 92% increase in accumulation in infarcted myocardium when compared to adjacent non-infarcted myocardium. Targeting to upregulated adhesion molecules on the endothelium provides a promising strategy for selectively delivering compounds to the infarct region of the myocardium using our liposomal-based drug delivery vehicle.

Myocardial Oxygenation in Infarcted Hearts Predicted by a Microvascular Transport Model

Chronic heart failure is most commonly due to ischemic cardiomyopathy with previous myocardial infarction (MI). Rebuilding lost myocardium to prevent heart failure mandates a neovasculature able to nourish new cardiomyocytes. Previously we have used a series of novel techniques adapted from field of tumor biology to directly measure the scar neovasculature at 1–4 weeks post-MI in rats following left coronary artery ligation and with a view towards its ability to deliver and exchange oxygen (AJP, 2005, 289(1):H108–H113). In this study, we have developed an anatomically and functionally realistic mathematical model of oxygen transport in cardiac tissue to help us in deciding what angiogenic strategies should be used to rebuild the neovasculature. The model is constructed to match the microvascular anatomy of cardiac tissue based on available morphometric image and is used to predict tissue hypoxia in the MI rat hearts. Our results indicated that our model predictions match experimentally measured tissue hypoxia values, and can be used to predict distributions of oxygen concentration in the hearts, which is mainly dependant on the density of perfused vessels in the heart tissue. From the minimal oxygen concentration needed by myocytes to maintain their normal function, we can calculate the number of perfused vessels needed in the heart to avoid hypoxia; this can guide our work in rebuilding vascular networks using targeted delivery of pro-angiogenic compounds to infarcted myocardium. Supported by AHA.

Use of a monoclonal antibody specific for activated endothelial cells to quantitate angiogenesis in vivo in zebrafish after drug treatment

We have recently generated a monoclonal antibody (mAb), Phy-V002, which specifically labels activated vascular endothelial cells (EC) in zebrafish. Here, we show that this mAb labels activated EC in newly formed vessels in vivo without staining mature vessels or other tissues. Using this mAb, drug effects on in vivo EC migration and vessel formation were visually assessed by whole-mount immunochemical staining in the transparent embryo. In addition, we have developed a quantitative microplate-based ELISA that measures EC proliferation in vivo after drug treatment. We have validated the quantitative in vivo ELISA using several antiangiogenic small molecules with different mechanisms of action which were added directly to the fish water. Some of these drugs, including: 2-methoxyestradiol, flavopiridol, paclitaxel, and genistein, are currently in clinical trials. We also injected large molecule drugs, including 3TSR and TSR2+KRFK, recombinant human antiangiogenic peptides of thrombospondin-1, a natural protein. To demonstrate that proangiogenic effects can also be assessed in zebrafish, we assessed effects of penicillamine and simvastatin, two proangiogenic compounds shown to stimulate vessel formation in rodents. Using whole-mount immunochemical staining with Phy-V002, inhibition of EC migration and inhibition or stimulation of vessel formation were visually observed for each compound. Next, using the quantitative in vivo angiogenesis ELISA, we generated dose-response curves for each compound. Compared to conventional assays, advantages of using zebrafish to assess drug effects on angiogenesis include: (1) a short assay time; (2) easy animal maintenance; (3) use of small quantities of drug; (4) single dosing; (5) a quantitative assay format; and (6) use of statistically significant number of animals per test.

An in vitro evaluation of the effects of homocysteine thiolactone on key steps of angiogenesis and tumor invasion

Homocysteine thiolactone is a highly reactive homocysteine derivative that can react easily with proteins. Protein homocysteinylation has been suggested as a possible mechanism underlying the pathological consequences of impaired homocysteine metabolism. Homocysteine inhibits key steps of angiogenesis and tumor invasion. It can be hypothesized that homocysteine thiolactone could mimic the described anti-angiogenic and anti-invasive effects of homocysteine. Therefore, we studied the effects of homocysteine thiolactone on different key steps of angiogenesis and tumor invasion, using model endothelial and tumor cell lines. This study demonstrates that homocysteine thiolactone, in high contrast to homocysteine, is not an anti-angiogenic compound. Furthermore, our results suggest that homocysteine thiolactone could behave as a pro-angiogenic compound.