Published online before print April 23, 2007, doi: 10.1161/HYPERTENSIONAHA.107.089409
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(Hypertension. 2007;50:219.)
© 2007 American Heart Association, Inc.
Sixth International Workshop on Structure and Function of the Vascular System |
Proinflammatory Profile Within the Grossly Normal Aged Human Aortic Wall
From the National Institute on Aging (M.W., J.Z., L-Q.J., G.S., G.P., R.M., E.G.L.), Baltimore, Md; and the Department of Cardiovascular Pathology (F.D.K., R.V.), Armed Forces Institute of Pathology, Washington, DC. Current addresses: Istituti di Ricovero e Cura a Carattere Scientifico Cardiovascolare (G.S.), Gruppo Multimedica, Milan, Italy; the Division of Biochemistry (G.P.), Department of Biomedical Sciences, School of Medicine, University of Sassari, Sassari, Italy; and the CVPath Institute, Inc (F.G.K., R.V), Gaithersburg, Md.
Correspondence to Mingyi Wang, Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Intramural Research Program, 5600 Nathan Shock Dr, Baltimore, MD 21224-6825. E-mail mingyiw{at}grc.nia.nih.gov
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Abstract |
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Studies in animal models demonstrate that angiotensin II and its downstream signaling molecules, that is, matrix metalloproteinases and monocyte chemoattractant protein-1, increase within the diffusely thickened intima of central arteries with aging. Whether such age-related changes occur within the human arterial wall is unknown. We harvested "grossly normal thoracic aortas" from 5 young (20±3 years) and 5 old white males (65±6 years) at necropsy, after death from traumatic causes. The intimae of older samples were markedly and diffusely thickened compared with younger intimae and contained increased levels of angiotensin-converting enzyme, angiotensin II, angiotensin II receptor type 1, matrix metalloproteinases 2/9, monocyte chemoattractant protein-1, and collagen I and III proteins. In situ activities of metalloproteinases 2/9 were also significantly enhanced within old, normal aortas. The thickened intima of older aortas also contained a 5-fold increase in the embryonic form of smooth muscle myosin heavy chain–labeled cells than that of younger aortas, and these fetal-type cells were colocalized with angiotensin II protein staining. The ability of isolated smooth muscle cells to invade an artificial basement membrane in response to a monocyte chemoattractant protein-1 gradient increased with age. Furthermore, angiotensin II increased the invasive capacity of young smooth muscle cells, and this effect was reduced by a metalloproteinase inhibitor or an angiotensin II receptor blocker. Thus, in the absence of lipid infiltration, the aged human aortic wall exhibits a proinflammatory profile that renders it a fertile substrate for the development of arterial disease, for example, atherosclerosis and hypertension.
Key Words: human • aging • arterial remodeling • Ang II • matrix metalloproteinase (MMP) • MCP-1
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Introduction |
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Both atherosclerosis and systolic hypertension increase exponentially in humans with advancing age after 40 years.1 It has been well documented that diffuse intimal medial thickening and exaggerated angiotensin (Ang) II signaling are implicated in hypertension and atherosclerosis.1–7 Furthermore, in humans, when the central arterial intimal medial thickening includes lipid infiltration and atherosclerotic plaques, it is a potent risk factor for the development of future clinical atherosclerotic events.6
Diffuse intimal–medial thickening of central arteries accompanies aging in numerous species, including humans.1,2,8–10 In rats and nonhuman primates in the absence of lipid deposition, the thickened intima contains numerous smooth muscle cells (SMC) and exhibits increased Ang II immunostaining and increases of Ang II downstream signaling molecules, including transforming growth factor (TGF)-ß1, matrix metalloproteinases (MMPs), and monocyte chemoattractant protein-1 (MCP-1).11–15 An age-associated increase in arterial nicotinamide-adenine dinucleotide phosphate oxidase activity and a reduction in NO bioavailability have also been observed in rats.3,16 This age-associated proinflammatory profile in the diffusely thickened arterial wall in animal models occurs in the absence of macrophage infiltration and is generated by smooth muscle and endothelial cells that become reprogrammed with advancing age.10 Thus, this diffuse increase in arterial intimal–medial thickening involving exaggerated Ang II signaling that accompanies aging in animal models is not, ipso facto, attributable to subclinical atherosclerosis, as it is presently defined, but rather is a manifestation of arterial remodeling that occurs over time with advancing age.
Little is known, however, about human central intimal aging in the absence of lipid accumulation or atherosclerotic plaques. Specifically, it is unknown whether a proinflammatory state promoted by exaggerated Ang II signaling also occurs within the human arterial wall with aging, in the absence of experimental and clinical evidence of atherosclerosis, as it does in animal models. In the present study, we examined the intima and media in postmortem samples of descending thoracic aortas of younger and older humans who did not succumb to cardiovascular disease. Specific aortic sampling sites were grossly normal and exclusive of fatty infiltration or atherosclerotic plaques. The invasive properties of isolated SMCs in vitro from other human donors were also examined. Our findings indicate that an age-associated proinflammatory profile of the aortic model manifests itself in the absence of fatty infiltration or atherosclerotic plaque in humans, similar to that observed in animal models. This finding has implications regarding the exaggerated risk of arterial disease in the elderly.
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Methods |
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Human Aorta Preparations
Segments of descending thoracic aortas were procured within 24 to 48 hours of patients dying from noncardiovascular deaths because of unnatural causes (homicide, suicide, or accident). Grossly normal intima and media of 10 white males, which were divided into 2 age groups (young: 20±3 years, n=5, and old: 65±6 years, n=5), were obtained at autopsy, over a 2-year period (July 2001 to August 2003), from the Department of Cardiovascular Pathology, Armed Forces Institute of Pathology. This research activity is designated EXEMPT (NO 3353) by the Office of Human Subjects Research (OHSR) and has been entered in the Office of Human Subjects Research database of the National Institutes of Health. The protocol was modified according to the methods described previously.17
Histological Analysis
Movat pentachrome staining for collagen and elastic content, oil red staining for lipid, and Toluidine blue staining for mast cells were performed according to the modified Luna’s histopathologic methods (American Histolab, Inc).
Immunohistochemistry
Immunostaining for Ang II, angiotensin-converting enzyme (ACE), angiotensin II receptor type 1 (AT1), MMP2, MMP9, MCP-1, CD68, a macrophage-specific antibody, 
-smooth muscle actin, and embryonic form of smooth muscle myosin heavy chain (SMemb), a marker for fetal type SMC,18 was performed according to the modification of methods described previously.11–13 The immunohistological analysis was performed via a computer imaging program, according to the instructions provided by the manufacturer (MetaMorph Imaging System, Universal Imaging Corp) using light microscopy.
Gelatin Zymography and Western Blots
Polyacrylamide gel electrophoresis gelatin zymography and Western blotting for ACE, AT1, MMP2, and MMP9 were performed according to the methods described previously.12,13 The primary antibodies used were anti-AT1 (1:1000), MMP2 (1:1000), MMP9 (1:1000), MCP-1 (1:1000), and SMemb (1:1000).
In Situ Zymography
To detect and localize in situ gelatinolytic activity, in situ zymography was performed according to the modified protocol described previously.11,12
Cultured Human SMC and Invasion Assay
Commercially available cultured human aortic SMCs, harvested from a 25-year-old white male (#2708, Cambrex) and from a 53-year-old white male (#1596, Cell Applications Inc) were used to study the invasion response of SMCs to MCP-1 according to the modified protocol described previously.19
Statistical Analysis
All of the results were expressed as the mean±SEM. Statistical comparisons for age differences were determined via Student’s t tests and for SMC invasion analysis via a 1-way ANOVA followed by a Bonferroni posthoc test. A P<0.05 was taken as statistical significance.
For additional details on materials and methods, including human aortic preparations, immunohistochemistry, immunofluorescence, and in vitro invasion assay, see the data supplement available online at http://hyper.ahajournals.org.
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Results |
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Age-Associated Thoracic Aortic Remodeling
Morphological analysis showed that diffuse aortic intimal thickness within specimens from the older donors increased by 9-fold compared with those from the younger donors (P<0.05; Table and Figure 1A, top). The medial thickness also increased with aging, but to a lesser extent (30%) than that of the intima–medial thickness in young and old samples, averaging 1867±167 µm and 2492 ±245 µm, respectively (P<0.05). The vast majority of cells within the intima stained positively for
-smooth muscle actin, a marker of SMCs (Figure 1A, middle). Most of these intimal SMCs also stained positively with an antibody to SMemb, a marker for the fetal-type SMC18 (Figure 1A, bottom). The number of fetal cells in the intima of older samples increased 
5-fold compared with that of younger samples (Table). Also, aging increased intimal collagen type I by 5.0-fold and type III by 5.6-fold (Figure 1B and Table). In addition, sporadic clusters of macrophages (CD68 stained cells) were more numerous within aortic walls of older versus younger samples (Figure 2A and Table). Mast cells were also occasionally detected by Toluidine blue staining in the intima of older but not younger samples (Figure 2B). These intimal mast cells demonstrated signs of activation and released a large amount of granules (Figure 2B, right). Notably, lipids were not detectable in these grossly normal aortas of either age group (Figure 2C).
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Assessment of Arterial Renin–Angiotensin System in Samples From Young and Old Human Aortas
Ang II immunofluorescence increased within the aortic wall of samples from older donors (Figure 3A); in the thickened intima, the Ang II fluorescence fraction increased 4-fold in the old compared with young aortas (Table). Immunostaining of the AT1 protein, a cognate receptor for Ang II, also increased within the older aortic wall, particularly within the intima (Figure 3B). Western blotting showed that an abundance of intimal AT1 protein contained in a given amount of intimal lysates increased on average, 
4-fold in older compared with younger subjects (Table). Strong staining of ACE protein, a key regulator of Ang II production, was present in the old intima (Figure 3C), and its expression level increased by 11-fold compared with that in younger intima, as determined by Western blots (Figure 3C and Table). Furthermore, double staining showed that intimal Ang II staining colocalized with either ACE (Figure 4A) or chymase staining (Figure 4B) in older aortas. Consecutive section staining showed that most chymase staining was located around or within the granules of mast cells (data not shown). Interestingly, SMemb and Ang II staining were colocalized within cells located within the intima of samples from older donors (Figure 4C).
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Aortic MMP Activity Increases Within the Older Aortic Wall
Immunohistostaining demonstrated that expression of MMP2 and MMP9 proteins, downstream signaling molecules of the Ang II cascade,12,20,21 also increase within the older aorta, predominantly in the intima, resulting in an MMP gradient across the aortic wall (Figure 5A). Western blot quantification showed that the abundance of MMP2 was increased by 3.8-fold and MMP-9 by 2.2-fold in old versus young subjects (Table). In situ zymography indicated that total gelatinase activity in the aortic intima was enhanced with aging (Figure 6A). Furthermore, polyacrylamide gel electrophoresis zymography of intimal lysates indicated that the combined activity of intimal gelatinases was increased by 2.2-fold in older versus younger samples (Figure 6B), and MMP2/9 and Ang II staining are colocalized within the thickened intima (Figure 5B). The protein levels of MMP2 and MMP9 within the media (Figure S1A) and total medial MMP activity in situ also increased in the aortic wall from older donors (Figure S1B).
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MCP-1 Increases Within the Aortic Wall From Older Donors
The expression of MCP-1, another downstream molecule of the Ang II signaling cascade,4,22,23 is also expressed to a greater extent within the older versus younger human aortic wall (Figure 7A). The increased MCP-1 within the old aorta resides predominantly within the intima, resulting in a markedly increased, age-associated intimal–medial gradient of the chemoattractant MCP-1 (Figure 7B). Interestingly, MCP-1 and Ang II staining within the thickened intima are colocalized (Figure 7C).
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Isolated SMCs in Culture
That intimal Ang II and SMemb double staining of intimal cells of samples from older donors (Figure 4C) suggests that Ang II may be involved in the phenotypic shift of SMCs to the fetal form. We tested this idea in vitro by exposing isolated human SMCs from commercial sources to Ang II. Indeed, immunohistochemical staining and Western blots demonstrated that Ang II treatment increased SMemb protein in older SMCs, and this effect was inhibited by [Sar1Gly8]-Ang II acetate hydrate, an AT1 receptor blocker (Figure S2).
In rats, chronic Ang II infusion increases aortic MMP2 protein and activity in vivo, and exposure of SMC in vitro to Ang II induces MMP2 expression and secretion.12 Similarly, Ang II increases aortic MCP-1 expression both in SMCs in vivo and in vitro.22,23 That Ang II and MMP staining are colocalized in the thickened human intima (Figure 5B) suggests that increased production of Ang II may also be linked the increase in MMP in the human aorta as well. Indeed, preliminary data indicate that Ang II induces both MMP2 and MMP9 protein expression in human SMCs, and this effect is abolished by [Sar1Gly8]-Ang II acetate hydrate, an AT1 antagonist (Figure S3).
Both MMPs and MCP-1 increase the capacity of SMCs to invade the extracellular matrix.12,13,19 The gradients of MCP-1 within the older human aortic wall (Figure 7B) might be expected to promote enhanced SMC invasion from media to intima. We, therefore, examined the invasion potential of isolated SMCs in response to an MCP-1 gradient before and after exposure to Ang II. Figure 8 showed that, as in SMCs from rats,12,13 the average invasive capacity of human SMCs in response to an MCP-1 gradient increased by 4-fold with aging. Ang II treatment significantly increased invasion of younger but not older SMCs. Notably, these Ang II–induced effects were substantially blocked by the MMP inhibitor GM6001 or by the AT1 receptor blocker [Sar1Gly8]-Ang II acetate hydrate.
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P<0.05, vs untreated young cells; 
P<0.05, vs Ang II–treated old cells. |
Discussion |
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The present study shows that, in aortic samples from older donors, the grossly normal human aortic wall, particularly the intima, in the absence of lipid infiltration, is diffusely thickened and manifests numerous fetal-like SMCs, an increase in sporadic clusters of macrophages, an occasional mast cell, and a large amount of collagen types III and I. Our results also show that the Ang II signaling cascade molecules, including ACE, AT1, MCP-1, and MMPs, increase within the arterial wall from older human donors as in animal models.11 Local Ang II is a product of the cascade reactions of angiotensinogen/Ang I/Ang II, governed by renin and ACE or chymase, within the cellular and extracellular matrix of the arterial wall.24 Renin is released from endothelial, macrophage/monocyte, and mast cells, which facilitates local formation of Ang I in an autocrine or paracrine manner.25–27 Previous studies have shown that angiotensinogen expression significantly increases in aortic SMCs from older compared with younger rats.28 The transcription, protein level, and activity of ACE also increase within the aortic wall from older rats and nonhuman primates.11,29,30 Thus, present and previous studies fully support the notion that the increased production of local Ang II within the aged arterial wall in humans is facilitated by an enhancement of both ACE- and chymase-dependent pathways.
AT1 transcription, protein abundance, and biological effects are increased in older versus younger rats.31,32 A chronic infusion of Ang II to young rats increases carotid medial thickening and intimal infiltration by SMCs, collagen deposition, and increased arterial MMP2 transcription, translation, and activation, as well as increased TGF-ß1 activity.12 These structural and molecular changes mimic those that occur in old, untreated rats.12 The present study shows that increases in Ang II and age-associated arterial remodeling in humans are accompanied by increased MMP2 activity, as in rats and nonhuman primates. In the present study, aortic MMP9 was also increased in specimens from older donors. Interestingly, an exposure of Ang II and its age-associated downstream molecules MCP-1, calpain, TGF-ß1, tumor necrosis factor-, and interleukin-1 to young SMCs increases MMP2 up to the level of old untreated SMCs.12–14,33–37 Our preliminary data (Figure S3) support that early passage untreated SMCs from older human donors secreted more MMPs than younger SMCs, as has been observed previously in rats.12–14
SMC infiltration of the intima observed in the present study in humans is consistent with previous studies in rats and nonhuman primates.11,12 Surprisingly, most of the intimal SMCs within the human aortic wall of old donors are fetal-like cells. A chronic infusion of Ang II into rats increases renal SMemb, which is also substantially inhibited by an AT1 blocker.38 We observed that exposure of SMCs from older donors to Ang II increases SMemb expression, which is abolished by the AT1 antagonist (Figure S2). Thus, Ang II treatment of SMCs in vitro reprograms those cells to express fetal myosin.
The present study shows that the invasive capability of older human SMCs is enhanced, as observed previously in aortic SMCs from older rats.12,13 Furthermore, exposure of Ang II to human, young SMCs in the present study increases their invasive capacity. MMP activity is required for invasion of cultured SMCs through a synthetic basement membrane barrier (Figure 8A).7,8,19 Both the present and previous studies12 show that the increase in Ang II-induced MMP activation in younger aortas of animal models is accompanied by MCP-1 expression in the aortic SMCs.13 In the present study, we observed that Ang II promotes SMC invasion in response to an MCP-1 chemoattractant gradient in human SMCs (Figure 8B), and this effect is substantially reduced by GM6001, an MMP inhibitor or by [Sar1Gly8]-Ang II acetate hydrate, an AT1 antagonist. Thus, these findings suggest that increased Ang II signaling via increased MMP activity and upregulated MCP-1 may play a pivotal role in the increased invasive capacity of SMCs within an older aortic wall.
During arterial aging, increased Ang II signaling and MMP2 activity are accompanied by elevated TGF-ß1 and TGF-ß receptor type-II.12–14,39 TGF-ß1 is a powerful profibrotic cytokine. Collagen deposition is increased diffusely within the aortic wall of older human donors, predominantly in the intima, as in animal models.11,12,39 The activation of latent TGF-ß1 leading to enhanced aortic SMAD (similar to mother against decapentaplegic) signaling and subsequently to an increase in fibronectin and collagen expression in the aged aortic wall depends on the concomitant age-associated increase in MMP-2 activity.39 Interestingly, chronic Ang II infusion increases MMP2, TGF-ß1 expression, and collagen production within the aortic wall in young rats to the level that occurs in old, untreated rats.11–15,39 Thus, the Ang II–induced activation of TGF-ß1 via MMP activation is a potential molecular mechanism for increased arterial fibrosis in older persons.
Perspectives
The results of the present study, taken together, indicate that cell and matrix proteins in the grossly normal-appearing aortic wall undergo age-associated reprogramming in humans, as in animal models. This age-related profile of the grossly normal aortic wall that evolves in humans and in animal models is proinflammatory and is manifested by intimal infiltration of fetal cells, increased production of Ang II signaling pathway molecules MMP and MCP-1, and enhanced activation of MMPs. The interaction of this proinflammatory profile that appears to be attributable to aging, per se, with other well-known human risk factors, for example, altered lipid metabolism, smoking, and lack of exercise, likely render the aging human artery fertile soil for facilitation of the initiation and progression of the quintessential arterial diseases of our society: atherosclerosis or hypertension.8–10,34 Thus, inhibition of Ang II signaling, or its downstream molecules MMP, MCP-1, and TGF-ß, via pharmacological or nonpharmacological therapies, emerges as a potential candidate to slow arterial aging and to reduce or prevent age-associated arterial disease that occurs in epidemic proportions among older persons.
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Acknowledgments |
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Sources of Funding
This research was supported by the Intramural Research Program of the National Institutes of Health, National Institute on Aging, and the Department of Cardiovascular Pathology, Armed Forces Institute of Pathology.
Disclosures
None.
Received February 20, 2007; first decision March 7, 2007; accepted March 30, 2007.
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