Ground Substance

GROUND SUBSTANCE

Surface tension aligns the components of a column of liquid of mixed ingredients, e.g. blood, so that each flows in its own layer. Ellis’ law assigns to the surface the layer with the lowest surface tension. Its low surface tension places lipid in crucial apposition to the blood-endothelial barrier. In health, the intact barrier effectively controls egress of this surface lipid through the barrier into the arterial wall. But when damaged, the inter-endothelium cement becomes depolymerised and permeable. Hypertension has been shown to be associated with such change (1) as evidenced by levels of von Willebrand factor, a marker of endothelial damage, and by fragmentation of endothelial tissue scattered in atheromatous plaques. The various forms of mechanical stress capable of localizing atherosclerotic lesions, whether experimental, in congenital anomalies, or in the usual human subject have been described previously (2).

Endothelial cells are separated one from another by intercellular cement that morphologically is in continuity with the ground substance of the arterial intima. The intimal ground substance is the substrate in which cholesterol is ultimately deposited in atherosclerotic plaques. Fig 1 (a) is a high power photomicrograph of early atherosclerosis in a guinea pig while Fig 1 (b) is a low power view of an advanced lesion. The red of lipid staining identifies cholesterol deposited homogeneously throughout the amorphous ground substance of the intima. In Fig 1 (a) are seen occasional lipid containing macrophages, widely separated from each other by lipid stained ground substance. There are no round lipid globules that would indicate a surface tension effect, and the lipid is incorporated in a sheet into the depolymerised ground substance.

These lesions were not induced by cholesterol feeding, and the cholesterol blood levels were only slightly above the normally low levels characteristic of guinea pigs. The atherosclerosis was induced by a scorbutogenic diet without other manipulation. Ascorbic acid is the specific essential for maintenance of collagen of which ground substance consists (3). This being the case, scurvy, which specifically targets the ground substance throughout the body, attacks the arterial ground substance. Conversely, ascorbic acid restores to normal those lesions of the ground substance induced by scurvy (3), unless scar formation prevents this.

Mechanical stress, whether compression (5) or stretch (6), determines the sites of damage by scurvy. Compression by the grasping surface of the hand localizes xanthoma tuberosum to the palms (Fig 2), standing localizes cholesterol deposits to the soles of the feet. Xanthomas of the elbows, and of the buttocks are likewise the result of skin compression. The stress of blood pressure overload that determines the arterial deposition of cholesterol in the usual case of atherosclerosis is the stretching force in arteries and is symbolized by ‘T’ in Laplace’s law (2).

Mechanical stress localizes the atherosclerosis associated with scurvy. The atherosclerosis has all the characteristics of human atherosclerosis, and is proven to be scurvy morphologically and biochemically. The ascorbic acid content of human arterial tissue at autopsy (7), is likewise depleted or absent at sites vulnerable to atherosclerosis (e.g. the carotid sinus), and this deficiency is correctable by oral ascorbic acid in the days before death. The adjacent unstressed segment of the same carotid artery preserves its normal ascorbic acid value. Thus it can be concluded that ascorbic acid is consumed to different degrees at different arterial sites in the same patient, the factor consuming it in atherosclerosis being mechanical.

Scurvy of arteries has all the morphological features of atherosclerosis (Fig 1 (a) & (b). Cholesterol is deposited in the intimal ground substance, and intimal hemorrhage, common in human atherosclerosis, is found in the absence of other hemorrhagic diatheses (8). Fig 3 is an intimal hemorrhage in a scorbutic guinea pig. Thrombosis is common in human scurvy (9), its sticky inter-endothelial cement being the basis for initiating clotting (10).

Conclusion #1: Scurvy induces atherosclerosis in guinea pigs. No other manipulation such as cholesterol feeding is involved, and in the process, cholesterol blood levels are marginally elevated above the normal low levels for this animal. The morphological characteristics are identical to human atherosclerosis. As in the human, cholesterol deposition occurs in plaques, there is intimal hemorrhage, and the stage is set for platelet adhesion and thrombosis by the stickiness of the inter-endothelial cement. Human arterial tissue is biochemically focally scorbutic at sites of mechanical stress, and this ascorbic acid depletion, detected at autopsy, is preventable by ante-mortem ascorbic acid.

Fig 4 (a) shows the absorption of a scurvy-induced atherosclerotic plaque in a guinea pig treated with ascorbic acid. Note the patches of lipid-free ground substance in the midst of a large plaque of cholesterol. The details have been described in another paper (11). Fig 4 (b) is the almost total removal of all cholesterol in another scorbutic animal treated with ascorbic acid. Only remaining are many tiny lipid globules, each with its meniscus, the tell-tale sign of restored surface tension. The ground substance is repolymerized, and the lipid has been extruded from it, prepared for removal by macrophages.

Fig 5 (a) compares with 5 (b) two arteriograms of the same femoral artery, performed three months apart. During the interval, the patient was treated with oral ascorbic acid 500 mg t.i.d. Note that human atherosclerosis is beginning to be absorbed from some of the plaques (12). In the main central trunk, the radius of the lumen has increased from 1.5 mm to 2.5 mm as a result of plaque resorption. This may seem trivial, but when these figures are inserted in the mathematical measurement of flow (flow is directly proportional to R⁴), an original flow of 5.06 increases to 39.06 per unit of time.

Conclusion #2: The atherosclerosis of guinea pig scurvy is reversible by ascorbic acid replacement. Serial arteriography demonstrates human atherosclerosis is likewise reversible with oral ascorbic acid resulting in a small increase of vessel radius that yields a great increase of flow.

A study of the incorporation of acetate C14 into cholesterol and fatty acids by surviving tissues of normal and scorbutic guinea pigs (13) demonstrated that the aorta of the guinea pig synthesizes cholesterol at a rate of one tenth that achieved by the liver. This was unchanged in scurvy. The cholesterol deposited in intimal ground substance in atherosclerosis associated with scurvy does not therefore seem to be due to increased cholesterol synthesis by the arterial wall. An accumulation of cholesterol could be attributed to decreased local cholesterol breakdown. However cholesterol degradation does not occur in arteries. Thus, by excluding other possibilities for its accumulation in the intima, the evidence points to blood cholesterol migrating into the intima, via damaged inter-endothelial cement, and by physicochemical binding becoming an atherosclerotic plaque. Only when polymerization is restored, is the gateway closed for further lipid deposition, and the previously deposited lipid extruded in the ground substance delivered to macrophages to carry to the liver. This is achieved by ascorbic acid replacement. Macrophage ingestion of lipid globules is surface tension dependent, the force acting externally in the way surface tension supports a needle floating on undisturbed water.

Conclusion #3: The mechanism of plaque formation in atherosclerosis is a physicochemical binding of cholesterol to intimal ground substance that has been damaged by mechanical stress. This damage takes the form of depolymerisation of the ground substance of the inter-endothelial cement rendering it sticky for platelet adhesion and also permeable for cholesterol migration into the arterial intima. Disposal of cholesterol from an atherosclerotic plaque requires the ground substance to again be polymerized so that the lipid may be extruded, allowed to assume its globular configuration under the influence of externally directed surface tension, and thus acceptable for macrophage consumption. Simultaneously repolymerization arrests atherogenesis.

The normal ground substance undergoes depolymerisation in scurvy (13). This makes the ground substance sticky (glue is depolymerised collagen) and releases glycoprotein into the blood (14). When ascorbic acid is administered in scurvy, the state of the normal ground substance is quickly restored and the glycoprotein levels fall to normal (15).

Release of glycoprotein into the blood is seen in a wide variety of stress, not only mechanical. Some examples are myocardial infarction, tumours, infection, trauma, rheumatoid arthritis (16). Many are acute. One routine autopsy study of tissue showed latent scurvy by chemical analysis in 20% cases (17).

Conclusion #4: Depolymerisation of ground substance with its implications is detectable by finding an elevation of serum glycoprotein in a wide variety of stress, mechanical and otherwise.

BIBLIOGRAPHY

1. Tse, W.Y., S.R.J., Maxwell, et al, J. Human hypertension, 8:843, 1994.

2. Willis, G.C., C.M.A.J., 70: 1-9, 1954.

3. Wolbach, S.B., Howe, P.R. Arch. Path. & Lab. Med., 1:1, 1926.

4. Willis, G.C., C.M.A.J., 69:17-22, 1953.

5. Follis, R.H., Arch. Path., 35: 579, 1943.

6. Daldorf, G., J. Exper. Med. 50: 293, 1929.

Fig 1 (a) is a high power view of the diffuse deposit of red stained lipid as a sheet in the ground substance of a guinea pig aorta rendered scorbutic. No cholesterol feeding or other interference was involved. Note the ground substance is relatively amorphous and that the three macrophages have phagocytosed lipid, but form an inconspicuous part of the entire plaque. Fig 1 (b) is a low power picture of an advanced atherosclerotic aorta in a scorbutic guinea pig.

Fig 2 In xanthoma tuberosum, of which this is a case, the cholesterol deposits are in the mechanically exposed palms of the hands, the mechanical stress being compression, rather than stretching. After three months of oral ascorbic acid, 500 mg t.i.d., the plaques were totally absorbed.

Fig 3 Shows an intimal hemorrhage in a scorbutic guinea pig.

Fig 4 (a) Ascorbic acid therapy has partially resolved a large atheromatous plaque in a guinea pig whose atheroma was scurvy-induced. There are several islands of red staining lipid with clear gaps between. The original ground substance is now clear in these gaps, where once there was advanced atherosclerosis. (b) The almost complete resolution of cholesterol from a scorbutic atheromatous plaque after ascorbic acid therapy.

Fig 5 (a) Note the degree of indentation the plaques in the centre of the angiogram make into the contrast medium, and match with the same plaques in (b) where the indentation has decreased, indicating the plaques have decreased in size. In the interval the patient had been treated with oral ascorbic acid 500 mg t.i.d. for three months.