Inter-university Cape Heart Research Unit

Research highlights

The role of genes in heart attacks and heart failure
The modern approach to gain insight into disease processes is via the manipulation of genes, with subsequent evaluation of the specific role candidate genes play in disease development.

The Interuniversity Cape Heart Unit uses genetic manipulation on numerous mouse models to try and enhance our understanding about ischaemic heart disease (heart attacks) and heart failure. These experiments may, in the future, result in improved therapy for these common diseases in South Africa.

The management of hypertension in the developing world
In collaboration with the South African Hypertension Society and with Dr Gaziano at the Harvard School of Public Health, we are beginning to define parameters to evaluate the cost-effectiveness of managing hypertension in the developing world. This body of work is already being incorporated into policy and strategy for both effective, and more cost efficient use of resources for the treatment of cardiac disease in South Africa.

Strengthening of the heart muscle
This body of work revolves around understanding how to enable the heart muscle to become more tolerant/ resistant to the noxious environment present when a person has a heart attack. This will prevent heart damage in the event of heart attacks and will improve both the quality and length of the patient's life.

This field is advancing rapidly and should result in improved therapies to prevent the death of heart tissue in response to a heart attack in the near future. Our research outputs (see below), show we are prominent in this area and are, we believe, making a significant contribution to this very important field.

Biocompatible polymeric superstructures for cardiovascular prostheses.
The experimental devices and methods used for the production of polyurethane vascular prostheses have been continuously improved in order to improve the consistency of grafts produced.

The above device was used to produce 6mm vascular grafts for an in-vivo surface modification study The goal of producing well-defined spherical pores in the graft wall was achieved by using specially manufactured spherical microbeads of 100 micrometers as a soluble porogen in a phase precipitation process. A custom built device was used for the accurate reinforcement of the grafts with elastic polyurethane fibres specially extruded and spun for this purpose. An example of a graft produced by this method is shown below.

The successful production of these prototypes is a significant step towardsattaining our goal of producing synthetic vascular prostheses that will heal to such an extent, when implanted in the body, that they will mimic all functions of a natural artery.

Surface modification of polymeric biomaterials
Two modification techniques were developed and applied to the surfaces of porous structures in an attempt to pacify the surfaces towards macrophage attack and to promote better healing in the body. The two methods were:

1. ionically adsorbed "bottlebrush" copolymer; and
2. covalently attached collagen (a biological substance isolated from calf skin).

The substances were immobilised on Dacron felt disks (a material commonly used in vascular reconstructions and other surgical applications) and porous polyurethane disks and experimental vascular prostheses (See CVR01). The samples were implanted in two animal models, and the results and implications are discussed in CRU06: Attenuation of the macrophage response to biomaterial through surface modifications.

Numerical simulation of a tissue engineered artery graft and heart valve.
Finite element methods and modelling is a mathematical engineering tool used to aid complex designs with the help of computers. Currently this tool has been used in the department to look at the development of a better graft.

A recently completed MSc project (August 1999) in the department looked primarily at the design of a porous graft structure and the use of windings to reinforce this structure. Having gone over this MSc report it had been noted that further development is required to reinforce the porous structure.

In order to accomplish this, the Finite Element Modelling Section has been considering the development of an adventitial type reinforcing to cover the porous structure like a sock. The modeling department has a new MSc student who completed numerous courses in this area in November 1999 and has started to look at modelling this adventitial type reinforcing.

Finite element methods uses a scientific approach in order to optimise feasible designs. They also rely heavily on mathematics to model various materials and the MSc student has focused recently on the mathematical modelling of hyper-elastic (very elastic) materials, which will constitute most of the materials used in the design.

The mathematical model is required to ensure that the computers material model behaves like those being physically tested. This model will be used for the elastic materials in the research and design of the adventitial sock.

This MSc research project using finite element analysis will also include the development, manufacture and testing of a feasible design, and is scheduled to be completed by December 2000.

Colocalisation of immunoglobulin binding sites and sites of early mineralisation in bioprosthetic heart valves - an ultrastructural study
Fibronectin has been identified as a key antigen in the acquired immune response to porcine bioprosthetic valve tissue in a rabbit immunisation model. This finding prompted us to attempt colocalisation of immunoglobulin binding and sites of early valve calcification.

Although we intend to examine colocalisation at an ultrastructural level in the final analysis, we have designed this study to allow for stepwise examination of this phenomenon, beginning with simple light microscopic examination of previously implanted tissue, using histochemical staining for calcium phosphate (von Kossa stain), together with protein-A immunogold and enzymatic immunohistology. This work was undertaken by an M.Sc student who recently left South Africa to pursue a medical degree in Canada.

Work to date involved familiarisation with histochemical, immunohistochemical and immunogold staining and the techniques of sample embedding and sectioning. This work is, however, critical in characterising the immune response to bioprosthetic tissue and will continue in earnest in the coming year.

Relevance of preformed natural xenoreactive antibodies to bioprosthetic valve implants
Preformed natural antibody is pivotal to the failure of xenogeneic grafts both in the clinical and in the experimental setting.

This study examines the relevance of preformed natural antibody in the pathophysiology of non-vascularised bioprosthetic valve constructs.

We have performed large animal implants of bioprosthetic valve constructs and have collected sera pre-operatively and at different time points post-operatively. We are currently in the process of performing Western-Blot analysis against bioprosthetic tissue extracts. The titre of pre-formed antibody responses will be correlated with post-implant titres, as well as against the pathophysiological outcome of the implanted valves. Antibody class will also be investigated.

This study was delayed due to the failure of an anticipated overseas student to join our group.

Attenuation of the macrophage response to biomaterials through surface modifications.
Foreign body inflammatory reaction towards implanted biomaterials is considered the nemesis of implanted medical devices. We therefore evaluated macrophage adhesion to surface modified polyurethane and Dacron biomaterials. Observations were made in vitro and in vivo in both the subcutaneous rat and baboon iliac models.

Four different surface modifications of two medical polymers were evaluated. Of these, modification with polyethylene glycol star polymers lead to significant reduction of macrophage adhesion to polymer surfaces in vitro. However, this trend was not observed in vivo.

In both rat and baboon models, pre-grafting with collagen was most effective at reducing the relative area of foreign body giant cell formation. No treatment eliminated giant cell formation completely.

Further investigations are necessary to determine what the impact of such observations may be. We are currently focusing on the effect of porous polymers on vascularisation.

Manipulating cell specific adhesion to extracellular matrix proteins to achieve selectivity: vascular endothelial cells versus smooth muscle cells
Migration studies using the confocal microscope with a migration stage have progressed towards positively identifying peptides that are either stimulatory or inhibitory towards migration of the crucial endothelial cells. This considerably strengthens the quest towards engineered tissue grafts that allow selective migration of targeted cells. The focus is now directed towards the second stage, investigating entirely synthetic matrices with shorter peptides, obtained from Professor Hubbell at the University of Zurich.

Three dimensional tissue engineered matrices potentiating cytokine-mediated vessel formation in vitro
Engineered fibrin matrices containing bound bioactive peptides have been obtained via continuing collaboration with Professor Hubbell at the University of Zurich. The microbead assay, after substantial fine tuning, has confirmed the angiogenic inhibitory properties of one of the peptides, strongly suggesting that the assay will be able to detect any of the stimulatory peptides required for the tissue engineered graft. In addition, progress has been made towards establishing an in vivo angiogenesis assay, which will be critical for confirming data obtained from the microbead assay.

Manipulating cell mediated matrix proteolysis to achieve selective tissue ingrowth into a 3D synthetic matrix
One approach to achieving selective population of an implanted biomaterial by a specific cell type is to incorporate selectively degradable matrices into the device. We have studied the secretion profile of matrix metalloproteinases by vascular and inflammatory cells using zymography and western blot techniques. We have established that under defined conditions vascular cells secrete higher amounts of stromelysin-1 (MMP-3) than macrophages. The peptide cleavage site of these enzymes could theoretically be incorporated into a degradable matrix to achieve preferential degradation by vascular, as opposed to inflammatory, cells.

Biocompatability and foreign body giant cell formation
Matrix metalloproteinases have been implicated in the failure of bioprosthetic heart valves as a result of tearing. We have studied the regulation of secretion of these extracellular matrix-degrading enzymes by unfixed and fixed fibronectin. Intact fibronectin molecules upregulated the secretion of fibronectin by peripheral blood mononuclear cells.

We aim to evaluate the effect of fibronectin fixation further on this regulation, as well as the effect of proteolytic degradation products of fibronectin. Negatively, regulators of MMP secretion could then be incorporated into cross-linked porcine tissue used for bioprostheses.

Characterisation of the immune response to heart valve bioprostheses
Previous research conducted by this Unit has shown the presence of a specific and reproducible acquired immune response to bioprosthetic valve tissue in a pig to rabbit immunisation model. In particular, fibronectin was identified as central to that response and work is still underway to characterise that finding further. The link between this response and its pathophysiological effects was uncertain and we therefore attempted to indicate the relevance of graft specific antibody in an implant model.

The New Zealand White rabbit was chosen as a recipient animal since the tissue specific sera had previously been generated in this animal. Porcine aortas were collected at the abattoir, punched into 6mm coupons and fixed according to a standard commercial regimen, but further detoxified to avoid cytotoxicity to immune competent cells.

Thereafter, the coupons were incubated with rabbit serum taken from previously immunised animals, either prior to the animal's initial immunisation or following immunisation and booster dosing with similarly fixed and detoxified tissue. Eight replicates were defined for each of five subsets of animals immunised.

Following serum incubation, the coupons were implanted subdermally in non-immunised New Zealand White rabbits. Upon explantation of the coupons three weeks later, the extent of calcification was determined by atomic absorption spectroscopy.

Results showed dramatic and elevated calcification in coupons incubated with graft specific sera from all but one of the five animals immunised, relative to coupons incubated with pre-immune control sera. This result clearly indicates the relevance of graft specific antibody in the pathophysiological process of bioprosthetic graft mineralisation.

Additional studies are underway to characterise the involvement of complement further and to rule out the effect of age differences between animals at the time of immunisation, and at the time of final immune sera collection.

Service to the sick
The clinical consultation service attends to patients with severe dyslipidaemia by providing a clinical assessment to the patient and such information is later available for research purposes and is viewed as relevant to our region and country. We see about 500 new patients per year and do about 1500 follow-up consultations. There is also an inpatient service for acute problems. The plasmaphoresis for homozygous familial hypercholesterolaemia is supervised from our clinic. This centre is the most experienced one in Africa in this regard.

The clinical consultation service of the Lipid Clinic attends to patients with severe dyslipidaemia by providing a clinical assessment to the patient. This information is later made available for research purposes and is relevant to both the Cape region and the country as a whole.

The Unit sees about 500 new patients per year, and does approximately 1500 follow-up consultations. There is also an inpatient service for acute problems.

The plasmapheresis for homozygous familial hypercholesterolaemia is supervised from our clinic, being the most experienced centre in Africa.

Patients were seen at Frere Hospital on an outreach programme from UCT.

Clinical investigation.
The Lipid Clinic evaluated two new drugs during the past year. Avasimibe was studied in extremely severe cases of hypercholesterolaemia. This agent may enhance the lipid-lowering effect of atorvastatin in homozygous familial hypercholesterolemia. A new statin study was also performed in homozygous familial hypercholesterolemia.

Laboratory investigation
A major research interest at our clinic is prevalence and causes of type III dyslipidaemia. We have previously found that a large proportion of local patients have an unusual molecular defect for this disorder, especially black patients.

We have set up a low cost screening test (non-denaturing gradient gel electrophoresis) which seems to detect the disorder well and could make a contribution to the diagnosis of patients with cholesterol disturbances world-wide.

We are also attempting to study the prevalence of this unusual gene for type III hyperlipidaemia, as it is a disease placing patients at very high risk for heart disease. Preliminary data suggest than an unusual mutation causing type III is prevalent in the Cape Town black population. This may have significant public health implications.