Role of matrix metalloproteinases in delayed cortical responses after stroke.

Matrix metalloproteinases (MMPs) are zinc-endopeptidases with multifactorial actions in central nervous system (CNS) physiology and pathology. Accumulating data suggest that MMPs have a deleterious role in stroke. By degrading neurovascular matrix, MMPs promote injury of the blood-brain barrier, edema and hemorrhage. By disrupting cell-matrix signaling and homeostasis, MMPs trigger brain cell death. Hence, there is a movement toward the development of MMP inhibitors for acute stroke therapy. But MMPs may have a different role during delayed phases after stroke. Because MMPs modulate brain matrix, they may mediate beneficial plasticity and remodeling during stroke recovery. Here, we show that MMPs participate in delayed cortical responses after focal cerebral ischemia in rats. MMP-9 is upregulated in peri-infarct cortex at 7-14 days after stroke and is colocalized with markers of neurovascular remodeling. Treatment with MMP inhibitors at 7 days after stroke suppresses neurovascular remodeling, increases ischemic brain injury and impairs functional recovery at 14 days. MMP processing of bioavailable VEGF may be involved because inhibition of MMPs reduces endogenous VEGF signals, whereas additional treatment with exogenous VEGF prevents MMP inhibitor-induced worsening of infarction. These data suggest that, contrary to MMP inhibitor therapies for acute stroke, strategies that modulate MMPs may be needed for promoting stroke recovery.

Functional muscle regeneration with combined delivery of angiogenesis and myogenesis factors.

Regenerative efforts typically focus on the delivery of single factors, but it is likely that multiple factors regulating distinct aspects of the regenerative process (e.g., vascularization and stem cell activation) can be used in parallel to affect regeneration of functional tissues. This possibility was addressed in the context of ischemic muscle injury, which typically leads to necrosis and loss of tissue and function. The role of sustained delivery, via injectable gel, of a combination of VEGF to promote angiogenesis and insulin-like growth factor-1 (IGF1) to directly promote muscle regeneration and the return of muscle function in ischemic rodent hindlimbs was investigated. Sustained VEGF delivery alone led to neoangiogenesis in ischemic limbs, with complete return of tissue perfusion to normal levels by 3 weeks, as well as protection from hypoxia and tissue necrosis, leading to an improvement in muscle contractility. Sustained IGF1 delivery alone was found to enhance muscle fiber regeneration and protected cells from apoptosis. However, the combined delivery of VEGF and IGF1 led to parallel angiogenesis, reinnervation, and myogenesis; as satellite cell activation and proliferation was stimulated, cells were protected from apoptosis, the inflammatory response was muted, and highly functional muscle tissue was formed. In contrast, bolus delivery of factors did not have any benefit in terms of neoangiogenesis and perfusion and had minimal effect on muscle regeneration. These results support the utility of simultaneously targeting distinct aspects of the regenerative process.

Supramolecular crafting of cell adhesion.

The supramolecular design of bioactive artificial extracellular matrices to control cell behavior is of critical importance in cell therapies and cell assays. Most previous work in this area has focused on polymers or monolayers which preclude control of signal density and accessibility in the nanoscale filamentous environment of natural matrices. We have used here self-assembling supramolecular nanofibers that display the cell adhesion ligand RGDS at van der Waals density to cells. Signal accessibility at this very high density has been varied by changes in molecular architecture and therefore through the supramolecular packing of monomers that form the fibers. We found that branched architectures of the monomers and the consequent lower packing efficiency and additional space for epitope motion improves signaling for cell adhesion, spreading, and migration. The use of artificial matrices with nanoscale objects with extremely high epitope densities could facilitate receptor clustering for signaling and also maximize successful binding between ligands and receptors at mobile three-dimensional interfaces between matrices and cells. Supramolecular design of artificial bioactive extracellular matrices to tune cell response may prove to be a powerful strategy in regenerative medicine and to study biological processes.

Sustained delivery of plasmid DNA from polymeric scaffolds for tissue engineering.

The encapsulation of DNA into polymeric depot systems can be used to spatially and temporally control DNA release, leading to a sustained, local delivery of therapeutic factors for tissue regeneration. Prior to encapsulation, DNA may be condensed with cationic polymers to decrease particle size, protect DNA from degradation, promote interaction with cell membranes, and facilitate endosomal release via the proton sponge effect. DNA has been encapsulated with either natural or synthetic polymers to form micro- and nanospheres, porous scaffolds and hydrogels for sustained DNA release and the polymer physical and chemical properties have been shown to influence transfection efficiency. Polymeric depot systems have been applied for bone, skin, and nerve regeneration as well as therapeutic angiogenesis, indicating the broad applicability of these systems for tissue engineering.

Cellular response to zinc-containing organoapatite: an in vitro study of proliferation, alkaline phosphatase activity and biomineralization.

We present a series of experiments investigating the in vitro biological activity of zinc-containing organoapatite (ZnOA)-coated titanium meshes. ZnOA is a hydroxyapatite-based material that contains poly(l-lysine) and zinc ions and can be coated onto titanium by treating the metal surface with poly(amino acids) that allow for electrostatic bonding of the mineral to the titanium surface. Preosteoblastic mouse calyaria cells were cultured on ZnOA-coated titanium meshes in a three-dimensional (3D) bioreactor, which provides an in vitro culture environment that better simulates what cells experience in vivo, compared to traditional 2D cultures. Results of these studies show a time-dependent cascade of events leading to an earlier onset of alkaline phosphatase (ALP) expression and biomineralization of ZnOA-coated samples as compared to controls. After the observation of peak ALP levels in ZnOA-coated titanium samples, mineralized bone nodules were observed by scanning electron microscopy. Tetracycline staining confirmed that the observed mineral nodules were newly synthesized biomineral, and not due to the inorganic coating. ZnOA-coated titanium substrates represent a new class of materials for human repair that provide, mechanical stability, as well as chemical and biochemical signals to promote new bone growth.

Self-assembling peptide amphiphile nanofiber matrices for cell entrapment.

We have developed a class of peptide amphiphile (PA) molecules that self-assemble into three-dimensional nanofiber networks under physiological conditions in the presence of polyvalent metal ions. The assembly can be triggered by adding PA solutions to cell culture media or other synthetic physiological fluids containing polyvalent metal ions. When the fluids contain suspended cells, PA self-assembly entraps cells in the nanofibrillar matrix, and the cells survive in culture for at least three weeks. We also show that entrapment does not arrest cell proliferation and motility. Biochemical and ultrastructural analysis by electron microscopy indicate that entrapped cells internalize the nanofibers and possibly utilize PA molecules in their metabolic pathways. These results demonstrate that PA nanofibrillar matrices have the potential to be used for cell transplantation or other tissue engineering applications.

A New Class of Supramolecular, Mixed-Metal DNA-Binding Agents: The Interaction of Ru(II),Pt(II) and Os(II),Pt(II) Bimetallic Complexes with DNA.

A new type of mixed-metal, supramolecular complex has been designed that incorporates a platinum center to allow binding to DNA. The interaction of two such platinum heterobimetallic complexes of the general formula [(bpy)(2)M(dpb)PtCl(2)]Cl(2) (M = Ru(II), Os(II); bpy = 2,2'-bipyridine; dpb = 2,3-bis(2-pyridyl)benzoquinoxaline) with DNA is reported herein. The modular design of these systems allows for synthetic variation of individual components within this structural motif. In this case, the remote metal is varied from Ru(II) to Os(II). DNA binding was analyzed using non-denaturing agarose gel electrophoresis. The interaction of these complexes with DNA was studied relative to the known DNA cross-linkers, cis-[Pt(NH(3))Cl(2)] (cisplatin) and trans-{[PtCl(NH(3))(2)](2)(&mgr;-H(2)N(CH(2))(6)NH(2))}(2+) (1,1/t,t). Our mixed-metal Ru,Pt and Os,Pt compounds retard the migration of DNA through the gel in both a concentration- and time-dependent manner. Their effect on the migration of DNA is similar to, although much more dramatic than, that observed for either cisplatin or 1,1/t,t. Our evidence suggests a covalent binding of our mixed-metal complexes to DNA through the platinum site. The degree of retardation of DNA migration suggests a large change in DNA conformation is induced by binding of our mixed-metal complexes. This work establishes these inorganic systems as a new class of DNA-binding agents and lays the groundwork for future efforts to enhance binding in an effort to develop novel anticancer drugs through serial design and testing.

Injectable hydrogels providing sustained delivery of vascular endothelial growth factor are neuroprotective in a rat model of Huntington's disease.

Vascular endothelial growth factor (VEGF) is a potent peptide with well-documented pro-angiogenic effects. Recently, it has also become clear that exogenous administration of VEGF is neuroprotective in animal models of central nervous system diseases. In the present study, VEGF was incorporated into a sustained release hydrogel delivery system to examine its potential benefits in a rat model of Huntington's disease (HD). The VEGF-containing hydrogel was stereotaxically injected into the striatum of adult rats. Three days later, quinolinic acid (QA; 225 nmol) was injected into the ipsilateral striatum to produce neuronal loss and behavioral deficits that mimic those observed in HD. Two weeks after surgery, animals were tested for motor function using the placement and cylinder tests. Control animals received either QA alone or QA plus empty hydrogel implants. Behavioral testing confirmed that the QA lesion resulted in significant deficits in the ability of the control animals to use their contralateral forelimb. In contrast, the performance of those animals receiving VEGF was significantly improved relative to controls with only modest motor impairments observed. Stereological counts of NeuN-positive neurons throughout the striatum demonstrated that VEGF implants significantly protected against the loss of striatal neurons induced by QA. These data are the first to demonstrate that VEGF can be used to protect striatal neurons from excitotoxic damage in a rat model of HD.