Studies in Cutaneous Aging: II. The Microvasculature

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1 X/82/ $02.00/0 THE JOURNAL OF lnvestigativ E DERMATOLOGY, 78: , 1982 Copyright ]982 by The Williams & Wilkins Co. Vol. 78, No.5 Printed in U.S.A. Studies in Cutaneous Aging: II. The Microvasculature IRWIN M. BRAVERMAN, M.D., AND EILEEN FONFERKO, B.A. Department of Dermatology, Yale University School of M edicine, New Haven, Connecticut, U.S.A. We studied by light and electron microscopy the microcirculatory vessels in the sun exposed and sun protected skin of normal and psoriatic individuals in order to separate the features of actinic damage from those of chronological aging. In actinic ally damaged skin, the vascular walls of postcapillary venules and of arterial and venous capillaries were thickened by the peripheral addition of a layer of basement membrane-like material. The veil cells which were intimately related to these layers often had dilated cisternae of rough endoplasmic reticulum containing electron dense material. In 3 of 8 individuals, 70, 70 and 72 yr old, the buttock skin showed mild vascular wall thickening. In 5 other patients, yr old the vessels of the buttock skin were normal. In 4 individuals yr old, the vessels were abnormally thin ( p.m). The veil cells were either absent or decreased in number in these specimens. We propose that (1) the veil cell is responsible for the synthesis and maintenance of the peripheral portion of the vascular wall of the dermal microcirculatory vessels; (2) the veil cell is stimulated to produce excessive basement membrane-like material in response to UV light, factors associated with diabetes mellitus, and possibly to factors associated with the early phases of chronological aging; and (3) with progressive aging there is a decrease in the number and synthetic activity of veil cells which correlates with the appearance of abnormally thin walled vessels. for study by light and electron microscopy by the techniques described in that report. These experiments were approved by the Human Investigations Committee at Yale University, School of Medicine. Light Microscopy RESULTS Thickened vessels could be recognized by the intensity and width of the PAS positive-diastase resistant staining of the vascular wall in both sun exposed and sun protected skin. The Laidlaw stain for }"eticulin fibers showed a poorly developed network around the vessels in the papillary dermis of buttock skin in 4 subjects who were found to have abnormally thin vascular walls by electron microscopy (see below). Electron Microscopy Extensor forearm shin: The walls of all the microcirculatory vessels in the upper dennis were thickened in 8 of 9 subjects, who were older than 58 yr, and exhibited cliriical signs of actinic damage on their extensor forearm skin. The postcapillary venules were more severely involved than the terminal arterioles In the preceding paper [1] we reported our observations on the elastic fiber abnormalities in actinically damaged and chronologically aged (sun protected) skin. In this report, we describe the changes in the microvasculature in these 2 sites. BACKGROUND The dermal arterioles, capillaries, and venules differ from the microcirculatory vessels of other organs in that their vascular walls are normally 2 to 3 }.lm thick [2]. By electron microscopy, the basement membrane material in the walls of arterial vessels has a homogeneous appearance, while in venous vessels, the basement membrane material is multilayered [2,3]. Each vessel in the dermis is surrounded by one or more flat fibroblast-like cells, called veil cells. Unlike the pericytes which are an integral component of the vascular wall and are surrounded by the mural basement membrane material, the veil cells are totally external to the wall (Fig 1). The veil cell demarcates the vessel from the surrounding dermis and can be considered to be an adventitial cell. The veil cells are infrequently seen around the microcirculatory vessels in the subcutaneous fat [4), and it is not known whether they are present around the microcircula-. tory vessels in other organs. MATERIALS AND METHODS The skin biopsies were obtained from the same sites and same patients described in the accompanying paper [11. They were processed Manuscript received June 26, 1981; accepted for publication November 15, This work was supported by DHEW grants AM and AG Reprint requests to: Irwin M. Braverman, M.D., Department of Dermatology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT FIG 1. Normal dermal venous capillary. V = veil cells. E = endothelial cell. P = pericyte surrounded by basement membrane material of vascular wall (x 5,460). 444

2 May 1982 THE MICROVASCULATURE IN CUTANEOUS AGING 445 FIG 2. Thickened postcapillary venule in extensor foreru'm skin. V = veil cells with dilated cisternae of rough endoplasmic reticulum. P pericytes. E = endothelial cell. B = layer of basement membrane-like material being deposited on periphery of vessel wall (x 4,550).

3 446 BRAVERMAN AND FONFERKO Vol. 78, No.5 biopsies from the buttocks, we found vessels with abnormally thin walls (Fig 6). These changes were confined primarily to the postcapillary venules. The venous and arterial capillaries were affe<;ted to a lesser degree. The arterioles were normal. The number of layers of basement membrane material in the wall was normal in some areas of the vessel and reduced to a single basal lamina between the endothelial cell and the Surrounding dermal collagen in other areas (Fig 6). The less dense basement membrane material normally present between the lamellae in the vascular wall was greatly diminished. The walls of these thin vessels measured approximately J.Lm (normal 2-3 J.Lm). The veil cells surrounding the thin vessels were either absent or fewer in number. They also appeared to be metabolically less active because there was either an absence or a reduction in the number of dilated cisternae in their cytoplasm. The 8 patients with vascular thickening in their extensor forearm skin, also had marked reduplication of the basal lamina at the dermal epidermal junction (DEJ). The reduplication consisted of 3 to 10 layers of lamina densa with their associated anchoring fibrils. The individual lamina densa was also irregularly thickened. The layers were often compacted, producing a false impression of pure basement membrane thickening as may be seen at the DEJ of diabetic skin. In 5 of the 8 subjects, a similar but less extensive thickening was found at the DEJ of their buttock skin. The biopsies from the normal and psoriatic subjects showed identical findings in these studies. FIG 3. Higher magnification of portion of vascular wall shown in Fig 2. V = veil cell with dilated cisternae. B = layer of basement membranelike material admixed with reticulin fibers on both sides of veil cell. Original vessel wall indicated by width of multilayered basement membrane material (arrows) (reduced from x 5,535). and capillaries. The thickening was not produced by a widening of the basement membrane material of the wall per se. Rather, the thickening was produced by a perivascular layer of basement membrane-like material admixed with individual collagen (reticulin) fibers (Fig 2 and 3) that was contiguous with the wall in some areas and separated from it in others. A veil cell or a portion of this cell was usually present in the space between the vessel and the newly deposited material. The veil cells appeared to be increased in number and they were usually in contact with, or in close proximity to, the deposited material. The basement membrane-like material was often present on both the adluminal and abluminal sides of the veil cell. The cytoplasm of the veil cells contained dilated cisternae of rough endoplasmic reticulum filled with electron dense matter (Fig 4). The thickness of the walls of affected vessels was increased by J.Lm. The majority of vessels were severely affected as illustrated in Fig 2 and 3; a minority of vessels showed mild thickening (Fig 5). In 6 persons who had normal appearing extensor forearm skin and who were under age 40, vascular wall thickening was not seen. An identical examination of the clinically normal flexor forearm skin from 12 healthy nondiabetic individuals aged 16 to 65 yr, who have served as controls in our electron microscopic studies, also failed to reveal vascular wall thickening. Buttock skin: Three of 8 individuals, aged 59 to 85 yr, with thick vessels in their extensor forearm biopsies, had moderate vascular wall thickening in their buttock skin similar to the pattern shown in Fig 5. We did not observe severe thickening as illustrated in Fig 2. These 3 subjects were. 70, 70, and 72 yr old. The other 5 patients had normal vessels. None of the 6 patients under age 40 yr showed vascular wall thickening in their buttock.skin. In 4 other subjects, 80 to 93 yr old, in whom we only had DISCUSSION The intimate relationship of the veil cells to the perivascular deposits, their increased numbers around thickened vessels, the presence of dilated cisternae containing electron dense material in these cells indicating synthetic activity, and the paucity of veil cells around abnormally thinned vessels suggest that these cells may be responsible for the observed changes in the vascular walls. The function of the veil cell around normal vessels is presently unknown, but it may be responsible for the synthesis of the basement membrane material admixed with reticulin fibers that comprir;;es the peripheral portion of the vascular wall of normal dermal vessels. The electron microscopic descriptions of vascular wall thickening in papillary vessels reported in erythropoietic protoporphyria, porphyria cutanea tru'da and variegate porphyria [5], as well as in lipoid proteinosis [6,7] appear to be identical to those we observed in actinically damaged skin. Kumakiri et al produced identical vascular changes in a volunteer who received 112 J/cm 2 ofuva irradiation every 2 weeks for almost 1 yr. Examination of their Fig 6 shows the presence of prominent veil cells within the areas of the perivascular deposits [8]. These controlled experiments with UV A irradiation provide additional evidence implicating UV light and veil cells in the production of the perivascular deposits of basement membrane-like material. The finding of thickened vessels with prominent veil cells in the buttock skin of 3 nondiabetic aged individuals, 70, 70 and 72 yr old, suggesting that this abnormality might also be a morphologic expression of aging. This hypothesis is supported by our observations of identical vascular changes in the buttock skin of 14 of 15 juvenile diabetics, 9-36 yr of age (unpublished data). These perivascular deposits are different from the true basement membrane thickening of capillary walls that has been observed and extensively studied in diabetic skeletal muscle. The abnormally thinned vessels in 4 subjects yr old were associated with a decrease in the number of veil cells as well as with a marked decrease or absence of metabolic activity as judged by morphology. Based upon the above observations, we propose that the veil cell, is stimulated by UV light, factors associated with diabetes mellitus and possibly by factors associated with chronological aging to produce vascular thickening by the perivasculru' deposition of basement membrane-like material. As aging pro-

4 FIG 4. Veil cell (V) with dilated cistel'llae of rough endoplasmic reticulum containing electron dense material. Identity of adjacent cell (N) is unknown, but probably is part of another veil cell. E = endothelial cell (x 9,225). FIG 5. Mild vascular wah thickening of arterial capillary in buttock skin of aged patient. Several veil cells (V) wi th dilated cistel'llae in intimate contact with new basement membrane-like material (B) being added to vasculru' wall. P = pericyte. E = endothelial cell (x 6,457).

5 448 BRAVERMAN AND FONFERKO Vol. 78, No ,.:.. :~,~. 'H m 4"-. : :~;::- 1 ~;.:; ~.;r. _ ~ ":_\1. ~.... / :~: "':.~;, ~- _~.. FIG 6. Postcapillary venule with abnormally thin wall in buttock skin of aged patient. Arrowheads indicate sites where vascular wall is reduced to a single basal lamina. Arrows indicate sites where multilaminations are present but the less dense basement membrane material normally present between the lamination is markedly decreased. Veil cell (V) is poorly developed. E = endothelial cell. P = pericyte (x 5,904). gresses, the functions of the veil cell begin to diminish, so that the basement membrane material of the wall is not replaced during normal metabolic turnover, and the vessel wall becomes thinner. The veil cell was first described by Rhodin in his study of the microvasculature in the subdermal fascia overlying the rabbit thigh muscle [9). In the rabbit, the veil cells are part of the adventitial connective tissue of postcapillary venules and muscular venules. In the human cutaneous microvasculature, they surround all the arterioles, capillaries and venules in the dermis, but are much less numerous in the subcutaneous fat [4). Available information about veil cells is meager. The postcapillary venule was the segment most affected either by vascular wall thickening or thinning. This segment has important physiologic functions not present in other parts of the microcirculation. It is the most permeable part of the microvasculature; diapedesis of white cells occurs here in response to a variety of stimuli; histamine, serotonin, and bradykinin act on this segment to produce increased vascular permeability; and circulating immune complexes are trapped in this.portion to produce vasculitis [2). Similarly, could the veil cells in this segment of the microvasculature be more reactive to stimuli than veil cells elsewhere in the microcirculatory system? The reduplication of basal lamina at the DEJ of buttock skin in 5 patients was similar but less extensive than that seen in the actinically damaged skin. Lavker observed the same reduplication of basal lamina at the DEJ of sun.protected and actinically damaged skin in his series of elderly patients [10). However, the significance of this alteration at the DEJ for the aging process remains to be determined. These changes may simply be a nonspecific response to a variety of epidermal injuries. In summary, we propose that the aging process has 2 major manifestations: elastic fiber abnormalities involving degradation and assembly, and vascular wall alterations of widening and atrophy depending upon the functional state of the veil cell. The finding of identical abnormalities in the skin of juvenile diabetics strengthens this hypothesis, as well as suggesting that these alterations are accelerated in diabetic patients. REFERENCES 1. Braverman 1M, Fonferko E: Studies in cutaneous aging. 1. The elastic fiber network. J Invest Dermatol, this issue 2. Yen A, Braverman 1M: Ultrastructure of the human dermal microcirculation: The horizontal plexus of the papillary dermis. J Invest Dermatol 66: , Higgins JC, Eady RAJ: Human dermal microvasculature: I. Its segmental differentiation. Light and electron microscopic study. Br J DermatoI104: , Braverman 1M, Keh-Yen A: Ultrastructure of the human dermal microcirculation. III. The vessels in the mid-and lower dermis and subcutaneous fat. J Invest Dermatol 77: , Epstein JH, Tuffanelli DL, Epstein WK: Cutaneous changes in t he porphyrias. Arch Dermatol 107: , Hashimoto K, K.lingmuller G, Rodermund O-E: Hyalinosis cutis et mucosae. An electron microscopic study. Acta Dermatovener (S tockh) 52: , Bauer EA, Santa-Cruz DJ, Eisen AZ: Lipoid proteinosis: In vivo and in vitro evidence for a lysosomal storage disease. J Invest Dermatol 76: , Kumakiri M, H ashimoto K, Willis I: Biologic cha nges due to longwave ultraviolet irradiation on human skin: ul trastructural study. J Invest Dermatol 69: , , Rhodin JAG: Ultrastructure of mammalian venous capillaries, venules, and small collecting veins. J Ultrastruct Res 25: , Lavker RM: Structural alterations in exposed and unexposed aged skin. J Invest Dermatol 73:69-66, 1979