Christian C. Haudenschild

Antiatherogenic effect of captopril in the Watanabe heritable hyperlipidemic rabbit.

The effects of 9 months of orally administered captopril (25-50 mg/kg body wt/day) on aortic atherosclerosis was examined in normotensive Watanabe heritable hyperlipidemic rabbits. Captopril caused a significant decrease in aortic atherosclerosis. Total aortic surface involvement by lesions was reduced from 48 +/- 3.6% in control Watanabe rabbits to 30 +/- 3.9% with captopril treatment (p less than 0.01). Most of the decrease could be accounted for by a marked reduction in atherosclerosis of descending thoracic aortas from 49 +/- 5.2% to 15 +/- 3.9% in control and captopril-related groups, respectively (p less than 0.001). Significant decrease in cholesterol content of descending thoracic aorta was also observed in captopril-treated rabbits. Microscopic examination of the arterial lesions in captopril-treated animals suggested a relative decrease in cellularity and increase in extracellular matrix as compared with untreated animals. These studies indicate that captopril has a potent antiatherosclerotic action in the Watanabe heritable hyperlipidemic rabbit.

Diabetes as an atherogenic factor

M ACROVASCULAR disease affecting large and medium sized arteries is the principal cause of mortality in diabetics in the western world.‘-5 Stroke, coronary heart disease, and especially peripheral arterial disease are more common in diabetics than in the general population and they occur at an earlier age (Table 1).677 In addition, diabetes substantially eliminates the relative protection possessed by women, and particularly by premenopausal women, from arterial disease.6’0 The principal cause of this arterial disease is atherosclerosis. I I-14 The acceleration of atherosclerosis in the diabetic is poorly understood although numerous mechanisms have been proposed.“-I9 The intent of this review is to examine some of these mechanisms, in light of cellular and extracellular events currently thought to be implicated in atherogenesis. Genetic, environmental, metabolic, and hormonal factors will be considered. In addition we will explore the possibility that atherosclerosis is not accelerated in all diabetics by a single mechanism; but that different factors predominate in different diabetic subgroups. The literature pertaining to the diabetes-atherosclerosis connection is extensive, and for this reason our approach will be somewhat selective. For a more comprehensive discussion of other aspects of this subject the reader is referred to the excellent reviews of Ganda,” Colwell et a1,16 and Jarrett et a1,13 and to two recent symposia.‘8*‘9

Recombinant platelet-derived growth factor B gene expression in porcine arteries induce intimal hyperplasia in vivo.

Platelet-derived growth factor (PDGF) B chain induces cell proliferation in vitro and is associated with arterial lesions that cause cardiovascular disease. However, it has been difficult to document the biological response to PDGF B gene expression in arteries in vivo. To determine the biologic effects of this growth factor in vivo, we have introduced an eukaryotic expression vector plasmid encoding recombinant PDGF B by direct gene transfer into porcine iliofemoral arteries using DNA liposome complexes. The presence of PDGF B plasmid DNA and expression of recombinant mRNA were confirmed by polymerase chain reaction analysis, and recombinant PDGF protein was demonstrated by immunohistochemistry. Intimal thickening was observed in porcine arteries 21 days following transfection with the recombinant PDGF B gene compared with arteries transduced with a control gene, E. coli beta-galactosidase. An eightfold increase in intimal to medial ratio was present in PDGF B gene transfected arteries compared with control transfected arteries (P = 0.001). This study suggests that expression of a recombinant PDGF B gene in vivo can play a role in the induction of intimal hyperplasia, which can lead to cardiovascular diseases.

Loss of proteolytically processed filaggrin caused by epidermal deletion of Matriptase/MT-SP1

Profilaggrin is a large epidermal polyprotein that is proteolytically processed during keratinocyte differentiation to release multiple filaggrin monomer units as well as a calcium-binding regulatory NH2-terminal filaggrin S-100 protein. We show that epidermal deficiency of the transmembrane serine protease Matriptase/MT-SP1 perturbs lipid matrix formation, cornified envelope morphogenesis, and stratum corneum desquamation. Surprisingly, proteomic analysis of Matriptase/MT-SP1–deficient epidermis revealed the selective loss of both proteolytically processed filaggrin monomer units and the NH2-terminal filaggrin S-100 regulatory protein. This was associated with a profound accumulation of profilaggrin and aberrant profilaggrin-processing products in the stratum corneum. The data identify keratinocyte Matriptase/MT-SP1 as an essential component of the profilaggrin-processing pathway and a key regulator of terminal epidermal differentiation.

Downstream anastomotic hyperplasia. A mechanism of failure in Dacron arterial grafts.

: The precise location and progression of anastomotic hyperplasia and its possible relationship to flow disturbances was investigated in femoro-femoral Dacron grafts in 28 dogs. In 13 grafts, the outflow from the end-to-side downstream anastomosis was bidirectional (BDO), and in 15 it was unidirectional (UDO) (distally). Grafts were electively removed at intervals of two to 196 days or at the time of thrombosis. Each anastomosis and adjacent artery was perfusion-fixed and sectioned sagittally. The mean sagittal section was projected onto a digitized pad, and the total area of hyperplasia internal to the arterial internal elastic lamina and within the adjacent graft was integrated by computer. The location of the hyperplasia was compared with previously established sites of flow separation and stagnation. The observation was made that hyperplasia is significantly greater at the downstream, as compared with the upstream, anastomosis in both groups (BDO = p less than 0.001 and UDO = p less than 0.001) (analysis of variance for independent groups). Furthermore, this downstream hyperplasia was progressive with time (BDO p less than 0.01) (UDO p less than 0.01); Spearman Rank Correlation. There was no significant increase in the extent of downstream hyperplasia where flow separation was known to be greater (BDO). Five grafts failed (three BDO, two UDO), as a result of complete occlusion of the downstream anastomosis by fibrous hyperplasia. Transmission electron microscopy showed the hyperplasia to consist of collagen-producing smooth muscle cells. Anastomotic hyperplasia is significantly greater at the downstream anastomosis, is progressive with time, and is the primary cause of failure of Dacron arterial grafts in this model. Quantitative analysis of downstream anastomotic hyperplasia may be a valuable measure of the biocompatibility of Dacron grafts.

Angiogenic activity as a marker of neoplastic and preneoplastic lesions of the human bladder.

Angiogenic activity has been assessed in biopsy specimens from 49 patients with transitional cell carcinoma, benign prostatic hypertrophy and vesicoureteral reflux. Over 95% of the specimens containing malignant and premalignant transitional epithelium stimulated capillary proliferation on rabbit iris. In contrast, less than 10% of normal tissues had this effect. Sixty-one per cent of specimens with inflammatory round cells were angiogenic but this was reduced to 10% by preincubation with rabbit antihuman lymphocyte serum. Cystitis cystica also induced capillary proliferation even if incubated with the antilymphocyte serum. Angiogenic capacity may be an early marker of preneoplastic and neoplastic lesions of the human bladder.

Heparin-binding growth factor 1 induces the formation of organoid neovascular structures in vivo.

One of the promises of modern molecular biology has been the opportunity to use genetically modified human cells in a patient to permanently restore inborn errors of metabolism. Although it has been possible to introduce genes into mammalian cells and to control their expression, it has proven difficult to introduce mammalian cells as carriers of the modified genetic information into hosts. The successful implantation of selective cells cannot be achieved without adequate vascular support, an essential step toward integration and reconstitution of a new biological function. Although a partial solution to this problem has been found by inducing specific site-directed neovessel formation using heparin-binding growth factor 1 (HBGF-1) adsorbed to a collagen matrix, these implants function for only a short period (weeks). We now report the formation of organoid neovascular structures using polytetrafluoroethylene fibers coated with collagen and HBGF-1 implanted in the peritoneal cavity of the rat. The organoid structures contained readily visible vascular lumina and nonvascular structures that resemble nerve tissue. It was also possible to demonstrate that the vascular system on the implant is continuous with the vascular tree of the host. This feature was used to demonstrate that the organoid structures are capable of sustaining the biological function of implanted normal rat hepatocytes over long periods of time (months) in the homozygous Gunn rat, thereby facilitating future applications involving the delivery of new genetic information.

Trandolapril inhibits atherosclerosis in the Watanabe heritable hyperlipidemic rabbit.

The effects of trandolapril (0.25 mg/kg body wt per 48 hours) on aortic atherosclerosis were examined in the Watanabe heritable hyperlipidemic rabbit treated from 3 to 12 months of age. Trandolapril caused a significant decrease in atherosclerotic involvement of the intimal surface of total aorta from 56.3 +/- 5.0% in control Watanabe rabbits to 35.0 +/- 4.1% with treatment (p less than 0.01). The largest reductions were observed in descending thoracic aorta where 21.8 +/- 5.7% of intimal surface was involved in the trandolapril-treated animals versus 54.4 +/- 7.7% in the control group (p less than 0.01). Significant decreases also occurred in ascending aorta/arch and abdominal aortic segments. Cholesterol content of descending thoracic aorta was also significantly reduced in the trandolapril-treated rabbits. The atherosclerotic plaques in aorta from trandolapril-treated rabbits appeared to contain less foam cells and relatively greater amounts of connective tissue than those from control animals. These studies indicate that trandolapril inhibits aortic atherosclerosis in the Watanabe heritable hyperlipidemic rabbit. The similarity in results between the current study and that using captopril suggests that the antiatherosclerotic action of trandolapril and captopril represents a class effect related to angiotensin converting enzyme inhibition.

Microvascular endothelium and pericytes: High yield, low passage cultures

SummaryCultured microvascular endothelial cells (MEC) have become a valuable model for studies of microvascular physiology and pathology. Most current techniques involve manual removal of undesirable cell types or cloning, require one to several months, and yield high population doubling level cultures derived from a relatively small sample of the original population. We have devised a technique to more rapidly produce larger numbers of MEC. This method provided primary cultures consisting predominantly of MEC within 1 wk. The technique involves selective aspiration of gray matter from the bovine cerebral cortex followed by homogenization, sieving, enzymatic dissociation, and then dense plating (104 to 105 vessel fragments/cm2) onto gelatin- or fibronectin-coated plastic. Typical yields were 0.1 to 0.5 × 106 fragments/g of aspirated gray matter. The optimal culture medium for these cells was 15% equine plasma derived serum, 20% conditioned medium, 2% retinal extract, 60% fresh medium, and 500 μg/ml heparin. Cells attached within 24 h, well-spread colonies were present within 1 to 2 d, and cultures approached confluence within 2 to 3 d. Alkaline phosphatase staining confirmed the microvascular origin of the material plated. Morphology, Factor VIII-related antigen staining and 1,1′-dioctacecyl-3,3,3′3,-tetramethyl-indocarbocyanine perchlorate acetylated low density lipoprotein uptake suggested that MEC predominated. Cultures could be passaged and additionally purified by sequential exposure to pancreatin and trypsin-EDTA. Pancreatin selectively removed MEC colonies leaving a relatively homogeneous pericyte population. The relative ease with which such cultures can be produced should facilitate the in vitro study of brain microvascular function and may also provide insights useful for growing MEC from other vascular beds.

Influence of hypertension on aortic atherosclerosis in the Watanabe rabbit.

The effects of one-kidney, one clip Goldblatt hypertension on aortic atherosclerosis have been studied in the Watanabe heritable hyperlipidemic (WHHL) rabbit. Renovascular surgery was performed on WHHL rabbits at 3 months of age, and the rabbits were followed for periods of 3–6 months. Aortic atherosclerosis was assessed by measurement of intimal surface involvement with atherosclerotic lesions, determination of aortic free and ester cholesterol content, and microscopic examination. Systolic blood pressure increased by approximately 40–60 mm Hg in the renovascular surgical group as compared with the sham-operated group, but body weight, heart rate, serum cholesterol, and serum triglyceride were unaffected. Aortic atherosclerosis was increased in the hypertensive rabbits, even after 2–3 months of hypertension. At 3 months after renovascular surgery, the aortic surface area covered by atherosclerotic disease averaged 77±4.4% in hypertensive as compared with 16±3.3 in control rabbits. At 6 months after surgery, the values were 62±8.2% and 30±5.3% in the hypertensive and control rabbits, respectively. The differences in surface involvement and cholesterol content as a result of hypertension were particularly prominent in the descending thoracic aorta. Atherosclerotic lesions in the descending thoracic and abdominal aortic regions of normotensive WHHL rabbits were localized primarily to the ostia of branch vessels, but in the hypertensive rabbits, the involvement was typically very diffuse. No major differences in the nature of atherosclerotic lesions of comparable size were apparent by light microscopy. The results indicate that hypertension accelerates atherogenesis in the WHHL rabbit and suggest that this model may be valuable for studying the mechanisms by which such acceleration is induced.