Researchers report on a new approach to treating ischemia based on the administration of nanofilaments carrying VEGF-mimetic peptides. The cell-free technology comprises self-assembling peptide amphiphile (PA) nanostructures that display high densities of the VEGF-mimetic peptides on their surface.
When tested in a mouse model of hind-limb ischemia the injectable nanofibers triggered an increase in microcirculation, which boosted tissue perfusion, functional recovery, and limb salvage. The team, from Northwestern University, Hannover Medical School, and the Chicago-based Institute for Bionanotechnology in Medicine, detail their findings in PNAS. Their paper is titled “Supramolecular nanostructures that mimic VEGF as a strategy for ischemic tissue repair.”
One of the major problems associated with using VEGF as a therapeutic approach to promoting angiogenesis in ischemic tissue is the inadequate retention of protein within the target zone, the researchers note. Because the protein is retained only for hours, repeated administration is likely to be necessary, significantly increasing costs.
Although a number of biomaterial-based approaches have been developed in an attempt to control the spatial and temporal delivery of VEGF, such strategies are still reliant on recombinant proteins, and some can only be administered via invasive surgical implantation. Meanwhile, recent work has demonstrated the therapeutic potential of a platform comprising self-assembling filamentous nanofibers formed from customizable peptide amphiphile (PA) molecules.
The PA consists of a hydrophobic alkyl segment covalently linked to a peptide that comprises both an amino acid sequence that drives self-assembly of the molecules into nanofibers and a customizable bioactive domain designed to interact with target proteins. The resulting nanostructures have dimensions similar to filamentous structures in the extracellular matrix and can form gel networks at low concentrations in aqueous media.
The Northwestern team exploited the features of this synthetic platform to develop PA-based nanofibers displaying a VEGF-mimetic epitope as a feasible alternative to protein VEGF therapies. They initially confirmed the ability of the VEGF PA nanofibers to phosphorylate VEGF receptor 1 and VEGF receptor 2 in human umbilical vein endothelial cells. VEGFR1 and VEGFR2 are the primary VEGF receptors implicated in angiogenic signaling. The results also showed that prolonged stimulation of the cells using VEGF PA led to increased cell numbers and better survival in serum-starved conditions.
The researchers then moved on to test the platform in a chicken chorioallantoic membrane (CAM) assay, which is an established in vivo angiogenesis model. When VEGF PA was coated onto a glass coverslip and applied to the CAM, there was a 229% increase in the blood vessel density over the next three days. In comparison, CAM treatment with the VEGF peptide alone led to a 139% increase in blood vessel density. The results could be visualized in terms of the density of blood vessels at the point of CAM stimulation, which radiated outwards.
A mouse hind-limb ischemia model was then used to evaluate whether VEGF-mimetic PA nanofibers could be used as therapy for ischemic disease. VEGF PA or control treatments were administered by an intramuscular injection three days after the induction of critical ischemia. Animals treated using VEGF PA demonstrated significantly less necrosis and improvements in active limb motor function at both day 21 and day 28 compared with animals treated with VEGF peptide, mutant PA, or saline.
Laser Doppler perfusion imaging (LDPI) showed that the nanofiber therapy led to markedly enhanced recovery of tissue perfusion at two weeks post-injury and at day 28. Encouragingly, histological tracking of fluorescently tagged PA and peptide in muscle tissue of the ischemic hind limb revealed that the PA is retained significantly longer than the peptide control. Examination of harvested ischemic limb tissue confirmed that VEGF PA was still present 28 days post treatment.
Physiological improvements in the VEGF PA-treated animals were accompanied by evidence of proangiogenesis, consistent with those observed in the CAM assay. “Overall, the improvements in tissue perfusion, limb salvage, motor function, and capillarization point to the therapeutic utility of VEGF PA for ischemic tissue repair,” the authors state.
The team carried out a second set of in vivo studies to compare the effects of VEGF PA therapy with recombinant VEGF therapy using VEGF165 protein. Control animals were again treated using saline injection. Both the VEGF PA and VEGF165 performed similarly in terms of the resulting LDPI perfusion ratio, but limb necrosis scores indicated that only the VEGF PA treatment led to significantly enhanced tissue salvage: There was a trend for improvement in the VEGF protein group, but this was not significant compared with control animals.
VEGF PA and VEGF protein therapy also both resulted in significantly improved limb motor function compared with the control. Importantly, the measure that was most affected by VEGF PA treatment was histological capillary density in the ischemic hind-limb muscle.
Treatment with VEGF PA resulted in significantly more capillaries in the hind limb than treatment with either VEGF protein or control, the researchers stress. “This dramatic effect in capillarization could result from the prolonged retention and activity of the VEGF PA in the muscle tissue compared to the VEGF protein.”
“We have demonstrated the use of bioactive and biodegradeable nanostructures as a strategy for therapeutic angiogenesis,” they conclude. “We were especially encouraged that the VEGF PA compared favorably to a high dose of recombinant protein. Though PAs are biodegradable by design and thus will be eventually broken down into natural products, they have been shown here to remain in the ischemic tissue for over two weeks after injection.
"This result is a substantial improvement when compared to the retention time reported for VEGF protein on the order of a few hours. The demonstrated efficacy suggests further consideration of these systems as an alternative therapy to protein-based strategies currently being evaluated for ischemic cardiovascular diseases.”