Coronary C-reactive protein distribution: its relation to development of atherosclerosis.

Two hundred and ninety-nine paraffin-embedded human coronary artery sections from 68 autopsies, both male and female and with various causes of death, were examined for distribution of C-reactive protein (CRP) using the indirect immunofluorescence technique and high-resolution confocal microscopy. The results demonstrate, for the first time, the existence of CRP in human coronary arteries, with evidence of CRP deposits being associated with lipids within in the artery walls. Grades of CRP immunoreactivity positively correlate with relative intimal thickness and negatively correlate with relative lumen size. It is suggested, therefore, that CRP may be related to the development of atherosclerosis and that the development of coronary atherosclerosis is associated with a relapsing inflammatory/necrotic process occurring within the coronary intima.

Plasma protein insudation as an index of early coronary atherogenesis.

Two hundred ninety-nine paraffin-embedded coronary artery blocks from 68 autopsy cases were serially sectioned. The blocks were selected to provide a range from normal through various stages of atherosclerosis, and sections were examined with the indirect immunofluorescence technique for intramural distribution of plasma albumin, fibrinogen, and immunoglobulin gamma (IgG). Cryostat-sections of 44 blocks from 22 of the same cases were examined with the same technique for distribution of apolipoprotein B. Alteration of protein insudation in the artery wall was a sensitive index of coronary atherogenesis. The sequence in which these proteins were involved in the initiation and development of early atherosclerotic lesions was analyzed by determining the average relative intimal thickness and relative lumen size that was associated with the first occurrence of altered insudation of each of these proteins. Results indicate that changed plasma albumin insudation is the earliest sign of a focal intimal lesion, and increasing albumin insudation shows the strongest association with intimal plaque growth. The other proteins tested showed altered insudation, in the order IgG, fibrinogen, apolipoprotein B. The results indicate that a progressive increase in permeability of the coronary artery endothelium occurs in the early stages of atherogenesis. Patterns of IgG localization provide evidence of both early systemic and subsequent local immune reactions being involved in atherogenesis. Altered albumin and apolipoprotein B insudation levels have stronger correlation coefficients with relative intimal thickness and relative lumen size than do those IgG and fibrinogen. The extremely high correlation coefficients shown by albumin emphasizes the importance of edema in determining plaque size and lumen stenosis.

Immunohistochemical study of intimal microvessels in coronary atherosclerosis.

Two hundred ninety-nine human coronary artery paraffin-embedded tissue blocks were examined for intimal microvessel invasion by probing for factor VIII-associated antigen with indirect immunofluorescence and high resolution confocal microscopy. The results obtained confirm that intimal microvessels originate in the adventitia and show that the richness of intimal microvessels is strongly positively correlated with intimal thickness and negatively correlated with relative lumen size. A number of plasma constituents were examined in serial sections. Comparison of immunofluorescence distribution patterns of these components with intimal microvessel distribution patterns reveals that intimal microvessels leak plasma albumin into artery walls, exude fibrinogen, and are associated with the build-up of plasma cells within atherosclerotic lesions. Therefore, intimal microvessels are demonstrated to play important roles in the development of atherosclerosis.

The coronary arteries in cases of cardiac and noncardiac sudden death.

Thirty-four cardiac and 22 sudden noncardiac deaths in men were examined with an injection, radiographic, and dissection autopsy technique to obtain as many coronary narrowings as possible for study. The narrowest sites in each of the major coronary vessels (LAD, LCX, and RCA) from each subject were identified histologically for analysis. The parameters studied were size of lumen, estimated as percentage of vessel cross-section, vs. the age of subject and the grades of chronic inflammatory cell infiltrate, of neovascularization, of intimal haemorrhage, and of pultaceous cholesterol-rich deposit in the wall. Genstat statistical analysis revealed that the significant explanatory variables for the reduction in arterial lumen were active inflammation and cardiac cause of death. Neither age nor cholesterol-rich deposits had significant explanatory power. A pathogenic role for inflammation may well be possible and efforts will be made in the future to investigate its etiology.

Wound contraction. Correlations between the tension generated by granulation tissue, cellular content and rate of contraction.

The tension generated in vitro by samples of granulation tissue taken from wounds at various times after wounding has been measured. Throughout the period when in vivo contraction was occurring with a constant linear rate of movement of the wound margins the in vitro tension produced by samples of the granulation tissue remained constant at approximately 80 mg/mm2. With cessation of contraction in vivo, the tension generated by the granulation tissue fell to 40 mg/mm2. Morphometric studies demonstrated that total cellularity paralleled the tension developed. However, the decrease in cellularity preceded by approximately 4 d the decrease in the tension developed. Strong correlation between the rate of contraction and the maximum thickness of the granulation tissue was demonstrated.

The contractile properties of wound granulation tissue.

A systematic examination of the contractile properties of wound granulation tissue is presented. Shortening of and tension generated by granulation tissue in the presence of 30 mM diphenhydramine HCl have been measured. Analysis of the stress (load per unit area)/strain (extent of shortening) results from isotonic shortening studies showed that over the range of 0 to 2.5% shortening there was an approximately linear relationship between stress and strain with a high modulus of elasticity. At lower stresses, wide variations in the amount of shortening occurred with little change in stress. Our interpretation of these findings is that diphenhydramine HCl caused an active shortening of the granulation tissue by 2.5% of its length and contractions greater than this were the result of secondary effects such as coiling and bending of the strips. It is shown that the granulation tissue would have to shorten by 2% once every 3 days to account for observed in vivo rates of contraction for large human wounds and once every 13 hr for rapidly contracting experimental rabbit wounds. The time course of the development of isometric tension by the granulation tissue is shown to be consistent with the proposal that each contractile cell contributes equally to the overall tension developed and that each cell is individually activated by a critical concentration of diphenhydramine HCl which is transported through the tissue by diffusion.

Pathological vascularization of the coronary intima.

Secondary vascularization was found in the walls of diseased coronary arteries from 36 individuals who died after sudden cardiac arrest. In the series presented here, coronary arteries of hospital patients dying from all causes also showed a 100% incidence of pathological neovascularization. A process linking neovascularization and chronic inflammation to an ultimate infectious aetiology is suggested.

Wound contraction. The effects of scab formation and the nature of the wound bed.

The effects of scab formation and the nature of the wound bed on the contraction of wounds have been investigated. Rabbit paraffin gauze dressed flank wounds with the panniculus carnosus intact and rat paraffin gauze dressed full skin thickness back wounds had constant linear rates of movement of the wound margins throughout the time of contraction. Rabbit undressed wounds with the panniculus carnosus retained or excised and rat undressed wounds showed phases of rapid movement, no movement and normal movement of the wound margins during the course of contraction. These were effects of scab formation, they were transient and had little or no effect on the overall rate of wound contraction. The behaviour of rabbit undressed wounds with the panniculus excised was similar to that of comparable wounds with the panniculus intact, except that healing was delayed by approximately 3 days. Very variable behaviour was observed with rabbit paraffin gauze dressed wounds with the panniculus excised.

Morphology of irradiated microvasculature: a combined in vivo and electron-microscopic study.

The purpose of this study was to establish a standardized experimental system to investigate the local effects of ionizing irradiation in the absence of systemic responses due to radiation. Twenty fully healed rabbit ear chambers in 13 rabbits were irradiated with single exposures of 7500 rads of beta-rays from a strontium-90 source. The irradiation resulted in an acute response, with cellular infiltration of the ear chambers, loss of vasomotion in arterioles, and vasodilatation. Over the weeks following irradiation, a gradual reduction in the number of blood and lymphatic vessels was noticed. A gradual reduction in the number of cells in the interstitium was also observed. Finally, only a few thin-walled blood vessels remained. Their endothelium contained no recognizable organelles and in places had disappeared completely, to expose the vascular basement membrane to the blood. These vessels were observed to be still flowing. No evidence either of capillary blockage due to swelling of endothelial cells or of platelet aggregation leading to thrombosis was obtained.

In vivo morphology and ultrastructure of thyroid autografts in rabbit ear chambers.

Partial thyroidectomies were performed in half-lop rabbits. Small pieces of thyroid tissue were grafted into fully healed Sandison--Clark rabbit ear chambers. Thyroid grafts were observed directly through the compound microscope and sequential observations were recorded on photographic films and video tapes, with grafts remaining undisturbed within their vascular bed. Circulation within grafts was established within 3-5 d post-operatively, and between the second and fourth week pulsatile lymphatics loaded with lymphocytes could be observed within the grafts. Moreover, initial cellular infiltration, epithelial cell necrosis and regeneration have been recorded. Clear spaces or droplets observed within the colloid in vivo are considered to correspond to histological features long considered to be artefacts. This technique allows a non-intrusive approach to the study of the thyroid gland and thus can be used in studies of in vivo thyroid physiology, pharmacology, pathology and therapy.

A systematic study of wound contraction in mammalian skin.

The phenomenon of wound contraction has been studied in the skin of rats, rabbits and guinea pigs with the aim of obtaining a standardized wound system to be used for biological assay of in vivo agonists and antagonists of granulation tissue contraction. The sex of the animal, the time of day of wounding, the size and the shape of the wounds all had no apparent influence on the wound contraction curves expressed as per cent of original area against time. The test animal's age and species did have a significant influence on contraction of skin wounds which by analogy with results obtained with rigid splints attached to the skin could be attributed to variations in the thickness, rigidity and fixation of the skin to deep tissues. The shape of the final scar was influenced by the position of the wound on the body surface. The major factor in this instance was considered to be tensile forces acting within the skin. Wound contraction curves in rats and rabbits showed three distinct phases, namely early closure, stationary and logarithmic closure. Guinea pigs lacked the early closure phase. The exact role of the panniculus carnosus in wound healing is considered to be worthy of detailed study.

Morphologic changes in arterial grafts in rabbit ears.

The morphology of auto- and allografted segments of rabbit central ear arteries was studied at various times after grafting. Autografts showed thrombosis only in the immediate postoperative period. Autografts developed intimal thickenings whose cellular elements at all stages were almost exclusively myointimal in type. Their medial smooth muscle cells were viable at all stages. Allografts frequently thrombosed within 8 weeks of grafting. Intimal thickenings that developed in the first 6 weeks in allografts mainly contained infiltrating hematogenous cells with few myointimal cells. Immune cells infiltrated all of the layers of allograft walls, and the smooth muscle cells of their medias showed increasingly severe degeneration and by 8 weeks had completely disappeared. In long established allografts, the intima was extremely thick and contained myointimal cells and fibrous tissue. Their medias were fibrosed. In long standing allografts, immune cell infiltration was no longer present. The thrombosis of allografted arteries that occurred within 8 weeks of grafting was related to immunologic events observed within the vessel grafts. Differences between myointimal cells and smooth muscle cells with regard to their morphology and orientation were identified. A possible origin of myointimal cells from endothelial cells is suggested.

In vivo observations on arterial micrografts.

Few investigations have been made to date on the in vivo behavior of blood vessel grafts. An experimental model is described which permits continuous monitoring of grafted arteries both by gross examination and by in vivo microscopy. Allografts and autografts of the central ear arteries of rabbits, 0.5 mm in diameter and 10 mm in length, were transplanted in rabbit ears and subsequently enclosed with transparent plastic discs on each surface of the ear in place of the reflected skin. Allograft segments showed decreased vasomotor activity by about 3 weeks after grafting, whereas the recipient arteries and autograft segments retained their activity. Reduced activity of allograft segments was confirmed by their poor responses to vasoactive drugs. Many allografts thrombosed due to immune reaction between 3 and 8 weeks after grafting. Allografts that survived this episode went on functioning as efficient conduits of blood for many months thereafter.

The aortic tunica media of the developing rat. I. Quantitative stereologic and biochemical analysis.

The tunica media of thoracic aortas from female rats in age from newborn to 12 weeks were analyzed quantitatively by using stereologic techniques in the electron microscope. Collagen, elastin, smooth muscle, myofilaments, Golgi apparatus, ergastoplasm, and surface to volume ratios were among those components quantified. These parameters were correlated with measurements of medial thickness, blood pressure, and tangenital tension in the wall and with biochemical estimates of collagen and elastin. A progressive increase in morphologically recognizable collagen in the media was in parallel with the increasing tangenital wall tension. Biochemical analysis indicated the presence of much soluble collagen in the early stages of development. Elastin was the major component contributing to increasing wall thickness. The elastin laminae were completely formed by 4 weeks of age, but further elastin was laid down as branches extending between medial cells in the aortas of animals between 2 and 12 weeks of age. Smooth muscle cells were ovoid in young animals and contained large amounts of ergastoplasma and Golgi apparatus, but very few myofilaments. With advancing age, these cells became irregular in outline, and their surface to volume ratios doubled. The myofilament volume increased greatly during development, associated with a decrease in "undifferentiated" cytoplasm, ergastoplasm, and Golgi apparatus. The maximal development of the latter two organelles was coincident with large increases in the connective tissue components.