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Lysosomes in Vascular Smooth Muscle Cells

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Abstract

The sections in this article are:

1 General Properties of Lysosomes
1.1 Definition
1.2 Physiological Functions of Lysosomes
2 Methods Used to Study Lysosomes
2.1 Biochemical Techniques
2.2 Morphological Techniques
3 Lysosomes in Vascular Smooth Muscle
3.1 Properties of Lysosomes in Aortic Smooth Muscle Cells
3.2 Lysosmal Alterations Associated with Vascular Diseases
4 Lysosomal Functions in Vascular Smooth Muscle Cells and Implications for Disease
4.1 Implications for Atherosclerosis
4.2 Implications for Hypertension and Diabetes Mellitus
4.3 Possible Implications of Autophagy
4.4 Insights from Inborn States of Lysosomal Deficiency
5 Conclusion
6 Addendum
Figure 1. Figure 1.

Schematic model of a lysosome depicting some of its key features, including the hydrolytic capacity to degrade a spectrum of biological molecules and the property of enzymic latency due to protective lipoprotein membrane. Only a few of the enzymes known to be present in lysosomes are listed here.

Adapted from de Duve
Figure 2. Figure 2.

Diagram illustrating the various forms of lysosomes and phagosomes of the vacuolar apparatus and the interactions of these structures with each other and with the cell membrane. Each cell type is believed to have one or more of the circuits shown, but not necessarily all. Crosses symbolize acid hydrolases.

From de Duve and Wattiaux
Figure 3. Figure 3.

Schematic representation of density gradient centrifugation, with layering of the sample on top. The two kinds shown are based on differences in the particle sedimentation coefficient (s) and density (ρP), respectively. Diagram at right illustrates histogram form of frequency distribution of particles or markers as a function of tube height. Such a diagram can be converted to show frequency distributions as a function of either sedimentation coefficient or of density. For details of calculation, see Reference .

From de Duve
Figure 4. Figure 4.

Density gradient equilibration of postnuclear supernatant from rabbit aortic smooth muscle cell homogenate. A: frequency‐density distributions (± SD) for various marker enzymes and for protein. B: frequency‐density distributions for eight hydrolases. Shaded area represents, over an arbitrary abscissa interval, the enzyme remaining in the starting layer. Note the distinct distribution patterns seen for the enzymes associated with various organelles.

From Peters et al.
Figure 5. Figure 5.

Effect that previously injecting rabbits with low‐density detergent (Triton WR‐1339) has on equilibrium density of aortic smooth muscle cell lysosomes. Broken lines give frequency‐density distributions obtained for control postnuclear supernatant aortic cell preparations; solid lines give frequency‐density distributions obtained for postnuclear supernatant aortic cell preparations from rabbits four days after intravenous injection of Triton WR‐1339. Note the simultaneous shift to lower densities (left) of four acid hydrolases that indicates their common intracellular localization. Distributions of cytochrome oxidase and proteins remain unchanged.

From Peters and de Duve
Figure 6. Figure 6.

Aortic smooth muscle cell from 10‐wk‐old rat incubated for acid phosphatase activity. Note the characteristic filaments (f), surface vesicles (v), and basement membrane (B). Dense enzyme reaction product (lead phosphate) is seen as black accumulations over lysosomes (Ly) and Golgi cisterna (G).

Adapted from Wolinsky, Goldfincher, et al.
Figure 7. Figure 7.

Density gradient equilibration of postnuclear supernatant from homogenates of isolated calf aortic cells, aortic explant cells, and subcultured aortic smooth muscle cells. Subcultured cells were brought to confluence in medium containing 10% fetal calf serum (10 FCS) and then cultured 1 additional wk in media containing 10 FCS, 2% calf serum (2 CS), 10% calf serum (10 CS), or 50% calf serum (50 CS). Graphs show frequency‐density distributions of four lysosomal acid hydrolases. Broken lines repeat enzyme distributions of isolated cell preparations for comparison; number of experiments are given in parentheses. Note the marked increases occurring in the densities of lysosomes. Extent of the density shift depends on the type of culture medium used when cells are established as subcultures.

From Fowler, Shio, and Wolinsky
Figure 8. Figure 8.

A: view of juxtanuclear region of calf aortic explant cell grown 3 wk in medium containing 10% fetal calf serum. B: view of juxtanuclear region of calf aortic smooth muscle cell subcultured 1 wk in medium containing 10% calf serum. C: view of juxtanuclear region of calf aortic smooth muscle cell subcultured 1 wk in medium containing 50% calf serum. More lysosomes are seen in all cultured smooth muscle cells than in cells in situ. Lysosomal appearances (Ly) change depending on the specific culture conditions used. N, nucleus; M, mitochondria; RER, rough‐surfaced endoplasmic reticulum; G, Golgi apparatus; f, filaments.

From Fowler, Shio, and Wolinsky
Figure 9. Figure 9.

Specific activities of marker enzymes for mitochondria (cytochrome oxidase), plasma membrane (5′‐nucleotidase), lysosomes (acid phosphatase, β‐glucuronidase, β‐galactosidase, N‐acetyl‐β‐glucosaminidase), and for protein and total cholesterol content of aortic cell homogenates isolated from control and atherosclerotic rabbits. Values (mean ± SE) are plotted against severity of atherosclerosis graded 0 to IV. There were 6–12 animals in each group.

From Peters et al.
Figure 10. Figure 10.

Influence of cholesterol‐rich diet on density of isolated rabbit aortic smooth muscle cells equilibrated in a Metrizamide density gradient. Graph shows distributions of cell constituents as a function of gradient volume. Density gradient is shown by the staircase (top). Broken lines give distributions of aortic cell preparations from controls; solid lines give distributions from cholesterol‐fed rabbits (grade III atheromatous aortas). Shaded area represents initial position of cells. A new population of low‐density cells greatly enriched in free and esterified cholesterol and in four lysosomal hydrolases appears in atheromatous aortas of cholesterol‐fed rabbits.

Adapted from Haley, Shio, and Fowler
Figure 11. Figure 11.

A: electron microscopic appearance of a typical low‐density rabbit aortic cell. Cytoplasm of this foam cell is filled with lipid deposits. B: at higher magnification these deposits appear to be in two forms, cytoplasmic lipid droplets (L) and membrane‐bounded vacuoles (V) containing debris. Vacuoles have been identified as lysosomes by cytochemical methods . Arrays of filaments (f) are scattered throughout the cytoplasm.

From Haley, Shio, and Fowler
Figure 12. Figure 12.

Influence of a cholesterol‐rich diet on the density of rabbit aortic smooth muscle cell lysosomes. Graphs show distribution patterns of enzymes as a function of gradient volume after density equilibration in sucrose density gradient depicted by staircase on top. Starting material was a postnuclear supernatant of isolated rabbit aortic cells homogenate, brought to a density of 1.26 and layered initially at outer edge of gradient (shaded area). Broken lines give distributions in control preparations; solid lines give distributions in preparation from a rabbit exhibiting grade IV atheroma as a result of cholesterol intake. Extensive shift to the left of five acid hydrolases indicates lower density of lysosomes. Significant change in the distribution pattern of total cholesterol was seen; distributions of protein, 5′‐nucleotidase, and cytochrome oxidase (not shown), however, were not changed.

From Peters and de Duve
Figure 13. Figure 13.

Lipid accumulation and acid phosphatase reaction product within lysosomes of aortic smooth muscle cells from three species. A: section of foam cell from aorta of rabbit rendered severely atheromatous by a cholesterol‐rich diet. Products of acid phosphatase reactivity are seen as dense crystalline deposits in lipid‐laden lysosomes (Ly) containing membranous debris. [From Shio et al. .] B: smooth muscle cell from nonhuman primate atherosclerotic lesion. Note fusion of reactive lipid‐filled lysosomes (Ly) bordered by a trilaminar unit membrane (arrows). [From Goldfischer, Schiller, and Wolinsky .] C: aortic smooth muscle cell from aorta of young human male. Lysosomes (Ly) contain homogeneous lipid droplets (L) of varying electron density in addition to enzyme reaction product. [From Coltoff‐Schiller, Wolinsky, and co‐workers .]

Figure 14. Figure 14.

Light micrographs of frozen sections of rat aortas incubated for acid phosphatase activity. A: section from normal rat. B: section from hypertensive rat. Lysosomes (arrows) are more abundant and darkly stained in the hypertensive vessel. Bar equals 10 μm.

From Wolinsky et al.
Figure 15. Figure 15.

Levels of hydrolases in thoracic aortic medias from control (C), diabetic (D), and insulin‐treated diabetic (I) rats. Streptozotocin‐induced diabetes lasted for 4 wk; insulin treatment was given for 1 wk. Each point represents pooled intima‐media strips from four aortas. Significance of differences between groups (nonpaired t test) is shown under arrows.

From Wolinsky et al. , by permission of the American Heart Association, Inc
Figure 16. Figure 16.

Light microscopic sections of thoracic aortas of rats; aortas were incubated for N‐acetyl‐β‐glucosaminidase activity. A: aorta of control rat. B: aorta of diabetic rat. C: aorta of insulin‐treated diabetic rat. Reactive lysosomes (arrows) are numerous in the control vessels, are markedly diminished in the diabetic, and are numerous again in the insulin‐treated diabetic. Bar equals 10 μm.

From Wolinsky et al. , by permission of the American Heart Association, Inc
Figure 17. Figure 17.

Electron micrograph of a smooth muscle cell from a section of rat aorta incubated in diaminobenzidine medium. The animal had been injected previously with horseradish peroxidase. Reaction product is located outside the cell and in small vesicles (arrows) along the cell surface.

From Coltoff‐Schiller, Wolinsky, et al.
Figure 18. Figure 18.

Model of transformation of smooth muscle cell to foam cell in rabbit aorta as a result of cholesterol feeding. Lipoproteins laden with cholesteryl esters are believed to infiltrate across vascular endothelium into the extracellular space and to enter smooth muscle cells by endocytosis. Endocytic vacuoles acquire digestive enzymes by fusion with lysosomes, thus becoming secondary lysosomes. Complete digestion of the material taken up leads to the clearing of the lysosomes that are ready to be recycled into a new digestive event lower left). Relative deficiency of lysosomal cholesteryl esterase causes retention of undigested cholesteryl esters within lysosomes, which become progressively overloaded with these deposits while the cell adopts foamy appearance (lower right).

From de Duve
Figure 19. Figure 19.

Electron micrograph that shows huge cytoplasmic lysosomes (Ly) in smooth muscle cells of formalin‐fixed aorta from a patient with Hurler's disease. Smooth muscle cells are identified (inset) by characteristic filaments (Mf), surface vesicles (v), and basement membrane (B).

From Goldfischer, Wolinsky, and co‐workers
Figure 20. Figure 20.

Diagrammatic representation of cellular mechanisms whereby increased delivery of materials to or decreased removal of materials from the lysosome could lead to intralysosomal accumulations.



Figure 1.

Schematic model of a lysosome depicting some of its key features, including the hydrolytic capacity to degrade a spectrum of biological molecules and the property of enzymic latency due to protective lipoprotein membrane. Only a few of the enzymes known to be present in lysosomes are listed here.

Adapted from de Duve


Figure 2.

Diagram illustrating the various forms of lysosomes and phagosomes of the vacuolar apparatus and the interactions of these structures with each other and with the cell membrane. Each cell type is believed to have one or more of the circuits shown, but not necessarily all. Crosses symbolize acid hydrolases.

From de Duve and Wattiaux


Figure 3.

Schematic representation of density gradient centrifugation, with layering of the sample on top. The two kinds shown are based on differences in the particle sedimentation coefficient (s) and density (ρP), respectively. Diagram at right illustrates histogram form of frequency distribution of particles or markers as a function of tube height. Such a diagram can be converted to show frequency distributions as a function of either sedimentation coefficient or of density. For details of calculation, see Reference .

From de Duve


Figure 4.

Density gradient equilibration of postnuclear supernatant from rabbit aortic smooth muscle cell homogenate. A: frequency‐density distributions (± SD) for various marker enzymes and for protein. B: frequency‐density distributions for eight hydrolases. Shaded area represents, over an arbitrary abscissa interval, the enzyme remaining in the starting layer. Note the distinct distribution patterns seen for the enzymes associated with various organelles.

From Peters et al.


Figure 5.

Effect that previously injecting rabbits with low‐density detergent (Triton WR‐1339) has on equilibrium density of aortic smooth muscle cell lysosomes. Broken lines give frequency‐density distributions obtained for control postnuclear supernatant aortic cell preparations; solid lines give frequency‐density distributions obtained for postnuclear supernatant aortic cell preparations from rabbits four days after intravenous injection of Triton WR‐1339. Note the simultaneous shift to lower densities (left) of four acid hydrolases that indicates their common intracellular localization. Distributions of cytochrome oxidase and proteins remain unchanged.

From Peters and de Duve


Figure 6.

Aortic smooth muscle cell from 10‐wk‐old rat incubated for acid phosphatase activity. Note the characteristic filaments (f), surface vesicles (v), and basement membrane (B). Dense enzyme reaction product (lead phosphate) is seen as black accumulations over lysosomes (Ly) and Golgi cisterna (G).

Adapted from Wolinsky, Goldfincher, et al.


Figure 7.

Density gradient equilibration of postnuclear supernatant from homogenates of isolated calf aortic cells, aortic explant cells, and subcultured aortic smooth muscle cells. Subcultured cells were brought to confluence in medium containing 10% fetal calf serum (10 FCS) and then cultured 1 additional wk in media containing 10 FCS, 2% calf serum (2 CS), 10% calf serum (10 CS), or 50% calf serum (50 CS). Graphs show frequency‐density distributions of four lysosomal acid hydrolases. Broken lines repeat enzyme distributions of isolated cell preparations for comparison; number of experiments are given in parentheses. Note the marked increases occurring in the densities of lysosomes. Extent of the density shift depends on the type of culture medium used when cells are established as subcultures.

From Fowler, Shio, and Wolinsky


Figure 8.

A: view of juxtanuclear region of calf aortic explant cell grown 3 wk in medium containing 10% fetal calf serum. B: view of juxtanuclear region of calf aortic smooth muscle cell subcultured 1 wk in medium containing 10% calf serum. C: view of juxtanuclear region of calf aortic smooth muscle cell subcultured 1 wk in medium containing 50% calf serum. More lysosomes are seen in all cultured smooth muscle cells than in cells in situ. Lysosomal appearances (Ly) change depending on the specific culture conditions used. N, nucleus; M, mitochondria; RER, rough‐surfaced endoplasmic reticulum; G, Golgi apparatus; f, filaments.

From Fowler, Shio, and Wolinsky


Figure 9.

Specific activities of marker enzymes for mitochondria (cytochrome oxidase), plasma membrane (5′‐nucleotidase), lysosomes (acid phosphatase, β‐glucuronidase, β‐galactosidase, N‐acetyl‐β‐glucosaminidase), and for protein and total cholesterol content of aortic cell homogenates isolated from control and atherosclerotic rabbits. Values (mean ± SE) are plotted against severity of atherosclerosis graded 0 to IV. There were 6–12 animals in each group.

From Peters et al.


Figure 10.

Influence of cholesterol‐rich diet on density of isolated rabbit aortic smooth muscle cells equilibrated in a Metrizamide density gradient. Graph shows distributions of cell constituents as a function of gradient volume. Density gradient is shown by the staircase (top). Broken lines give distributions of aortic cell preparations from controls; solid lines give distributions from cholesterol‐fed rabbits (grade III atheromatous aortas). Shaded area represents initial position of cells. A new population of low‐density cells greatly enriched in free and esterified cholesterol and in four lysosomal hydrolases appears in atheromatous aortas of cholesterol‐fed rabbits.

Adapted from Haley, Shio, and Fowler


Figure 11.

A: electron microscopic appearance of a typical low‐density rabbit aortic cell. Cytoplasm of this foam cell is filled with lipid deposits. B: at higher magnification these deposits appear to be in two forms, cytoplasmic lipid droplets (L) and membrane‐bounded vacuoles (V) containing debris. Vacuoles have been identified as lysosomes by cytochemical methods . Arrays of filaments (f) are scattered throughout the cytoplasm.

From Haley, Shio, and Fowler


Figure 12.

Influence of a cholesterol‐rich diet on the density of rabbit aortic smooth muscle cell lysosomes. Graphs show distribution patterns of enzymes as a function of gradient volume after density equilibration in sucrose density gradient depicted by staircase on top. Starting material was a postnuclear supernatant of isolated rabbit aortic cells homogenate, brought to a density of 1.26 and layered initially at outer edge of gradient (shaded area). Broken lines give distributions in control preparations; solid lines give distributions in preparation from a rabbit exhibiting grade IV atheroma as a result of cholesterol intake. Extensive shift to the left of five acid hydrolases indicates lower density of lysosomes. Significant change in the distribution pattern of total cholesterol was seen; distributions of protein, 5′‐nucleotidase, and cytochrome oxidase (not shown), however, were not changed.

From Peters and de Duve


Figure 13.

Lipid accumulation and acid phosphatase reaction product within lysosomes of aortic smooth muscle cells from three species. A: section of foam cell from aorta of rabbit rendered severely atheromatous by a cholesterol‐rich diet. Products of acid phosphatase reactivity are seen as dense crystalline deposits in lipid‐laden lysosomes (Ly) containing membranous debris. [From Shio et al. .] B: smooth muscle cell from nonhuman primate atherosclerotic lesion. Note fusion of reactive lipid‐filled lysosomes (Ly) bordered by a trilaminar unit membrane (arrows). [From Goldfischer, Schiller, and Wolinsky .] C: aortic smooth muscle cell from aorta of young human male. Lysosomes (Ly) contain homogeneous lipid droplets (L) of varying electron density in addition to enzyme reaction product. [From Coltoff‐Schiller, Wolinsky, and co‐workers .]



Figure 14.

Light micrographs of frozen sections of rat aortas incubated for acid phosphatase activity. A: section from normal rat. B: section from hypertensive rat. Lysosomes (arrows) are more abundant and darkly stained in the hypertensive vessel. Bar equals 10 μm.

From Wolinsky et al.


Figure 15.

Levels of hydrolases in thoracic aortic medias from control (C), diabetic (D), and insulin‐treated diabetic (I) rats. Streptozotocin‐induced diabetes lasted for 4 wk; insulin treatment was given for 1 wk. Each point represents pooled intima‐media strips from four aortas. Significance of differences between groups (nonpaired t test) is shown under arrows.

From Wolinsky et al. , by permission of the American Heart Association, Inc


Figure 16.

Light microscopic sections of thoracic aortas of rats; aortas were incubated for N‐acetyl‐β‐glucosaminidase activity. A: aorta of control rat. B: aorta of diabetic rat. C: aorta of insulin‐treated diabetic rat. Reactive lysosomes (arrows) are numerous in the control vessels, are markedly diminished in the diabetic, and are numerous again in the insulin‐treated diabetic. Bar equals 10 μm.

From Wolinsky et al. , by permission of the American Heart Association, Inc


Figure 17.

Electron micrograph of a smooth muscle cell from a section of rat aorta incubated in diaminobenzidine medium. The animal had been injected previously with horseradish peroxidase. Reaction product is located outside the cell and in small vesicles (arrows) along the cell surface.

From Coltoff‐Schiller, Wolinsky, et al.


Figure 18.

Model of transformation of smooth muscle cell to foam cell in rabbit aorta as a result of cholesterol feeding. Lipoproteins laden with cholesteryl esters are believed to infiltrate across vascular endothelium into the extracellular space and to enter smooth muscle cells by endocytosis. Endocytic vacuoles acquire digestive enzymes by fusion with lysosomes, thus becoming secondary lysosomes. Complete digestion of the material taken up leads to the clearing of the lysosomes that are ready to be recycled into a new digestive event lower left). Relative deficiency of lysosomal cholesteryl esterase causes retention of undigested cholesteryl esters within lysosomes, which become progressively overloaded with these deposits while the cell adopts foamy appearance (lower right).

From de Duve


Figure 19.

Electron micrograph that shows huge cytoplasmic lysosomes (Ly) in smooth muscle cells of formalin‐fixed aorta from a patient with Hurler's disease. Smooth muscle cells are identified (inset) by characteristic filaments (Mf), surface vesicles (v), and basement membrane (B).

From Goldfischer, Wolinsky, and co‐workers


Figure 20.

Diagrammatic representation of cellular mechanisms whereby increased delivery of materials to or decreased removal of materials from the lysosome could lead to intralysosomal accumulations.

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Stanley Fowler, Harvey Wolinsky. Lysosomes in Vascular Smooth Muscle Cells. Compr Physiol 2011, Supplement 7: Handbook of Physiology, The Cardiovascular System, Vascular Smooth Muscle: 133-160. First published in print 1980. doi: 10.1002/cphy.cp020206