Enhanced neointimal growth in cultured rabbit aorta following in vivo balloon angioplasty

Summary We have used in vivo balloon catheterization in combination with in vitro organ culture to develop a model system for vascular neointima formation. A Fogarty balloon catheter was used to deendothelialize and rupture the internal elastic lamina of aortae in adult rabbits. After three d of recovery, aortae were harvested, divided into segments, and placed into organ culture. We obtained a daily index of cell proliferation in cultured vessels using [³H]thymidine incorporation into DNA. Also, segments were collected and processed for routine histology or immunohistochemistry. Aortic segments that had undergone ballooning 3 d before harvest and then cultured exhibited diffuse neointimal growth after several d in vitro, whereas those from sham-operated (nonballooned) rabbits showed generally only a single endothelial cell layer that is characteristic of normal intima. Aortae that were harvested, balloon-damaged in vitro, and then cultured exhibited no neointimal growth. The neointima that developed in cultured segments from in vivo ballooned rabbits was primarily of smooth muscle cell origin as determined by positive immunostaining for α-smooth muscle actin. The intima:media thickness ratios were significantly higher in aortic segments from ballooned rabbits at harvest and after 4 or 7 d in culture compared with those from nonballooned rabbits. Also, the [³H]thymidine index was higher in the in vivo ballooned aorta compared to non-ballooned or in vitro ballooned vessel. We conclude that ballooning in vivo followed by exposure to blood-borne elements produces an enhanced proliferative response in cultured vessels that is distinct from other in vitro models of neointimal growth.     

Novel model of inflammatory neointima formation reveals a potential role of myeloperoxidase in neointimal hyperplasia

Atherosclerosis, which is characterized by neointima formation, is an inflammatory disease. However, there is no inflammatory product-elicited neointimal model to support the causal role of inflammation in atherogenesis. We reported previously that leukocyte-derived MPO induces vascular injury responses such as endothelial dysfunction. We now test the role of MPO in inflammatory neointima formation. We infused temporarily isolated rat common carotid arteries with MPO (200 nM) and incubated for 1 h. We found that although MPO itself did not induce any neointima formation 2 wk after treatment, in the presence of its substrate, hydrogen peroxide, MPO was able to elicit neointimal hyperplasia. We further confirmed that MPO-induced neointimal hyperplasia is mediated by its product, hypochlorous acid (HOCl). HOCl elicited apoptosis both in intima and media followed by vascular proliferative response and resulted in neointima formation with a heterogeneous cell population. Both histological and functional features of HOCl-treated vessels are similar to those in atherosclerotic lesions. To our knowledge, this is the first direct in vivo demonstration of neointimal formation induced by a product of the inflammatory cascade. The results suggest that MPO may be a mediator for pathological neointima growth. This novel neointimal model could be useful for studying inflammation and atherosclerosis.     

Bradykinin receptor antagonists attenuate neointimal proliferation postangioplasty

Bradykinin has been linked to the development of restenosis in response to vascular injury. We therefore examined the effect of bradykinin on vascular smooth muscle cell growth and neointimal formation in organ culture. Bradykinin stimulated both RNA and DNA synthesis (by 175%) in smooth muscle cells from either porcine or human coronary arteries and increased cell number in a concentration-dependent manner. Both p42/44 mitogen-activated protein kinase (MAPK) and p38 kinase were also activated. Treatment with [Hyp(3),Tyr(Me)(8)]bradykinin, a B(2) receptor agonist, stimulated thymidine incorporation by 146%, whereas B(1)-selective Lys-des-Arg(9)-bradykinin had no effect. Addition of the B(2) antagonist HOE-140 reduced the stimulation by 56%, whereas B(1)-selective des-Arg-HOE-140 had no significant effect. Similarly, HOE-140 attenuated angioplasty-induced neointimal formation in organ culture with an efficacy approaching 100% inhibition. These experiments suggest that bradykinin promotes smooth muscle proliferation after vascular injury, presumably via B(2) receptor-dependent activation of MAPK family pathways, and may explain the negative outcome of angiotensin converting enzyme inhibitor therapy on restenosis in nonrodent models.     

Contribution of recipient-derived cells in allograft neointima formation and the response to stent implantation

Allograft coronary disease is the dominant cause of increased risk of death after cardiac transplantation. While the percutaneous insertion of stents is the most efficacious revascularization strategy for allograft coronary disease there is a high incidence of stent renarrowing. We developed a novel rabbit model of sex-mismatched allograft vascular disease as well as the response to stent implantation. In situ hybridization for the Y-chromosome was employed to detect male cells in the neointima of stented allograft, and the population of recipient derived neointimal cells was measured by quantitative polymerase chain reaction and characterized by immunohistochemistry. To demonstrate the participation of circulatory derived cells in stent neointima formation we infused ex vivo labeled peripheral blood mononuclear cells into native rabbit carotid arteries immediately after stenting. Fourteen days after stenting the neointima area was 58% greater in the stented vs. non-stented allograft segments (p = 0.02). Male cells were detected in the neointima of stented female-to-male allografts. Recipient-derived cells constituted 72.1+/-5.7% and 81.5+/-4.2% of neointimal cell population in the non-stented and stented segments, respectively and the corresponding proliferation rates were only 2.7+/-0.5% and 2.3+/-0.2%. Some of the recipient-derived neointimal cells were of endothelial lineage. The ex vivo tagged cells constituted 9.0+/-0.4% of the cells per high power field in the stent neointima 14 days after stenting. These experiments provide important quantitative data regarding the degree to which host-derived blood-borne cells contribute to neointima formation in allograft vasculopathy and the early response to stent implantation.     

HCMV-infection in a human arterial organ culture model: effects on cell proliferation and neointimal hyperplasia

Background

The impact of infections with the human cytomegalovirus (HCMV) for the development of atherosclerosis and restenosis is still unclear. Both a clear correlation and no correlation at all have been reported in clinical, mostly serological studies. In our study we employed a human non-injury ex vivo organ culture model to investigate the effect of an in vitro permissive HCMV-infection on cell proliferation and neointimal hyperplasia for a period of 56 days.

Results

During routine-nephrectomies parts of renal arteries from 71 patients were obtained and prepared as human organ cultures. Cell free HCMV infection was performed with the fibroblast adapted HCMV strain AD169, the endotheliotropic strain TB40E, and a clinical isolate (AN 365). After 3, 7, 14, 21, 28, 35, and 56 days in culture staining of HCMV-antigens was carried out and reactive cell proliferation and neointimal thickening were analysed. Successful HCMV-infection was accomplished with all three virus strains studied. During the first 21 days in organ culture no cell proliferation or neointimal hyperplasia was detected. At day 35 and day 56 moderate cell proliferation and neointimal hyperplasia was found both in HCMV-infected segments and mock infected controls. Neointimal hyperplasia in productively HCMV-infected segments was lower than in non infected at day 35 and day 56, but relatively higher after infection with the endotheliotropic TB40E in comparison with the two other strains.

Conclusion

The data do not support the hypothesis that HCMV-infection triggers restenosis via a stimulatory effect on cell proliferation and neointimal hyperplasia in comparison to non infected controls. Interestingly however, even after lytic infection, a virus strain specific difference was observed.     

Evidence for a role for a uterine renin-angiotensin system in decidualization in rats.

Experiments were performed in vivo and in vitro to determine the effects of enalaprilat, a specific inhibitor of angiotensin-converting enzyme, on various aspects of the decidual cell reaction in rats. Ovariectomized, adult female rats were sensitized for the decidual cell reaction with steroid treatments. For in vivo experiments, intrauterine infusions of enalaprilat alone, and in combination with angiotensin II and prostaglandin E2 (PGE2), were initiated on the day of uterine sensitivity. Enalaprilat inhibited the increases in uterine PG concentrations, endometrial vascular permeability, alkaline phosphatase activity and uterine weight that occurred sequentially following infusion of vehicle. Concurrent infusion of angiotensin II did not reverse any of these inhibitory effects; PGE2 infusion partially, but not completely, reversed the inhibition of increase in uterine weight, although it did not alter the inhibition of endometrial vascular permeability. For in vitro experiments, endometrial stromal cells were obtained from uteri on the day of sensitivity and cultured for up to 3 days in the presence of enalaprilat and angiotensin II. Enalaprilat inhibited in a dose-dependent manner the increases in stromal cell alkaline phosphatase activity and media PGE concentration that occurred in the control cultures; these effects were fully reversed by concurrent treatment with angiotensin II. The inhibition of stromal alkaline phosphatase activity was also reversed by PGE2; conversely, the ability of angiotensin II to reverse the effect of enalaprilat was lost in the presence of indomethacin. These studies provide evidence of a requirement for angiotensin II during the decidual cell reaction in rats and suggest that it acts, at least in part, through a PG-dependent mechanism.     

Optimisation and validation of an angiotensin-converting enzyme inhibition assay for the screening of bioactive peptides

Angiotensin-converting enzyme (ACE) plays a major role in the regulation of blood pressure. A diagnostic assay to measure angiotensin-converting enzyme (ACE) activity was transformed into an enzyme inhibition assay and optimised, which led to a more sensitive and less expensive assay. By this spectrophotometric method, ACE inhibition is measured using the substrate furanacryloyl-Phe-Gly-Gly and as ACE source rabbit lung acetone extract. The optimised as well as the original ACE inhibition assay were used to verify the ACE inhibitory activity of captopril. The ACE inhibition assay was further validated by enalapril, its active derivative enalaprilat and the ACE-inhibitory peptide Ala-Leu-Pro-Met-His-Ile-Arg, corresponding to a tryptic fragment of bovine beta-lactoglobulin. Sigmoid curves could be fit adequately to the data points representing ACE inhibition in function of inhibitor concentration. IC(50) values for these compounds corresponded well with literature data. Furthermore, pea and whey protein hydrolysates obtained by digestion with trypsin showed ACE inhibitory activity in the ACE inhibition assay. Hence, this optimised assay is suitable to screen for ACE inhibitory peptides derived from food proteins with a possible antihypertensive effect in vivo.     

Effects of peptidase inhibitors on bradykinin-induced bronchoconstriction in the rat

Objectives of this study were to determine if aerosolized bradykinin causes bronchoconstriction in anesthetized, mechanically ventilated rats, and if pretreatment with enalaprilat, an inhibitor of angiotensin-converting enzyme (ACE), or phosphoramidon, an inhibitor of endopeptidase 24.11 (EP 24.11), alters the response. We found that aerosolized bradykinin elicited a reproducible bronchoconstrictor response that was significantly amplified by pretreatment with aerosolized enalaprilat or phosphoramidon. Neither inhibitor alone affected airway tone or caused nonspecific airway hyperreactivity. These findings indicate that both ACE and EP 24.11 contribute to bradykinin degradation in rat airways.     

Smooth muscle cells isolated from the neointima after vascular injury exhibit altered responses to platelet-derived growth factor and other stimuli

A variety of evidence suggests that vascular smooth muscle cells (SMC) exhibit a more immature phenotype when stimulated by injury to replicate in the adult. One growth characteristic common to immature (embryonic, fetal, and neonatal) SMC is a markedly reduced responsiveness to platelet-derived growth factor (PDGF) and other mitogenic stimuli. Here we demonstrate that SMC isolated from the 14-day neointima of experimentally injured carotid arteries exhibit a similar growth phenotype. The proliferative responses of neointimal cells to the BB homodimer of PDGF, which interacts with both forms of the PDGF receptor, were up to twenty-fold less (as assessed by BrdU immunocytochemistry) than that of adult control tunica media cells over a wide range of PDGF concentrations. Paradoxically, these cells expressed abundant mRNA for the α- and β-subunits of the PDGF receptor (by RT-PCR) and expressed abundant PDGF receptor protein (by Western blotting). Addition of PDGF-BB to neointimal SMC induced significant autophosphorylation of the PDGF receptor, suggesting that the PDGF receptors were fully functional. The chemotactic responses of neointimal SMC to PDGF, in in vitro migration assays, were identical to that of control medial cells. The data further establish the existence of vascular SMC phenotypes characterized by a refractoriness to growth stimulation by specific mitogens, and provide further evidence for the reiteration of developmentally regulated programs following vascular injury in vivo. © 1996 Wiley-Liss, Inc.     

Pigment epithelium-derived factor reduces the PDGF-induced migration and proliferation of human aortic smooth muscle cells through PPARγ activation

Our previous study demonstrated that pigment epithelium-derived factor (PEDF) plays an important role in the proliferation and migration of human aortic smooth muscle cells (HASMCs). In the present study, we examined whether PEDF inhibited platelet-derived growth factor (PDGF)-stimulated HASMC migration and proliferation. PEDF dose-dependently reduced PDGF-induced HASMC migration and proliferation in vitro and also arrested cell cycle progression in the G0/G1 phase, and this was associated with decreased expression of cyclin D1, cyclin E, CDK2, CDK4, and p21(Cip1) and increased expression of the cyclin-dependent kinase inhibitor p27(Kip1). The antiproliferative and antimigratory effects of PEDF were partially blocked by the PPARγ antagonist GW9662, but not by the PPARα antagonist MK886. In in vivo studies, the femoral artery of C57BL/6 mice was endothelial-denuded and the mice injected intravenously with PEDF or vehicle. After 2 weeks, both the neointima/media area ratio and cell proliferation (proliferating cell nuclear antigen-positive cells) in the neointima were significantly reduced and again these effects were partially reversed by GW9662 pretreatment. Our data show that PEDF increases PPARγ activation, preventing entry of HASMCs into the cell cycle in vitro and reducing the neointimal area and cell proliferation in the neointima in vivo. Thus, PEDF may represent a safe and effective novel target for the prevention and treatment of vascular proliferative diseases.     

Tetrahydrobiopterin deficiency exaggerates intimal hyperplasia after vascular injury

Decreased levels of tetrahydrobiopterin (BH4), an absolute cofactor for nitric oxide synthase (NOS), lead to uncoupling of NOS into a superoxide v. nitric oxide producing enzyme, and it is this uncoupling that links it to the development of vascular disease. However, the effects of in vivo deficiency of BH4 on neointimal formation after vascular injury have not been previously investigated. Hph-1 mice, which display 90% deficiency in guanine triphosphate cyclohydrolase I, the rate limiting enzyme in BH4 synthesis, were used. Hph-1 and wild-type mice, treated with either vehicle or BH4 (n = 15 per group), were subjected to wire-induced femoral artery injury, and NOS expression and activity, inflammation, cell proliferation, superoxide production, and neointimal formation were assessed. The major form of NOS expressed over vessel wall after vascular injury was endothelial NOS. Hph-1 mice exhibited lower NOS activity (2.8 +/- 0.3 vs. 4.5 +/- 0.4 pmol/min/mg protein, P < 0.01), and higher aortic superoxide content (5.2 +/- 2.0 x 10(5) cpm vs. 1.6 +/- 0.7 x 10(5) cpm, P < 0.01) compared with wild-type controls, indicating uncoupling of NOS. Treatment of hph-1 mice with BH4 significantly increased NOS activity (from 2.8 +/- 0.3 to 4.1 +/- 0.4 pmol.min(-1).mg protein(-1), P < 0.05), and attenuated superoxide production (from 5.2 +/- 2.0 x 10(5) cpm to 0.8 +/- 0.7 x 10(5) cpm, P < 0.05). Hph-1 mice also had higher inflammatory reactions and more cell proliferation after vascular injury. Furthermore, hph-1 mice responded by a marked increase in neointimal formation at 4 wk after vascular injury, compared with wild-type controls (intima:media ratio: 4.5 +/- 0.5 vs. wild-type 0.7 +/- 0.1, P < 0.001). Treatment of hph-1 mice with BH4 prevented vascular injury-induced increase in neointimal formation (intima:media ratio: 1.4 +/- 0.1 vs. hph-1, P < 0.001). Treatment had no effect on wild-type controls. In summary, we describe, for the first time, that in vivo BH4 deficiency facilitates neointimal formation after vascular injury. Modulation of BH4 bioavailability is an important therapeutic target for restenosis.     

Transient reexpression of an embryonic autonomous growth phenotype by adult carotid artery smooth muscle cells after vascular injury

High rates of vascular smooth muscle cell (SMC) replication are observed, at least transiently, after injury to the arterial wall and contribute to the formation of a neointima. Neutralizing antibodies designed to inhibit growth of SMC have only been variably successful in inhibiting neointima formation, raising the possibility that neointimal cell proliferation involves unique growth mechanisms. This study examined the possibility that SMC isolated from injured rat carotid arteries would express an autonomous, mitogen-independent growth phenotype similar to that utilized by embryonic vascular SMC during periods of rapid growth. We found that primary cultures of SMC isolated 7 and 14 days after injury, times at which high in vivo replication rates were observed, demonstrated high intrinsic DNA synthetic rates compared to SMC isolated from uninjured arteries or at 2, 4, 21, and 28 days after injury where in vivo replication rates were far less. Subcultured SMC isolated from 7-day injured vessels (Neo7 SMC) exhibited a stable, autonomous growth phenotype, did not secrete detectable mitogenic activity, and had decreased alpha-actin and myosin expression compared to mitogen-dependent SMC. Heterokaryons constructed between autonomous Neo7 SMC and mitogen-dependent SMC exhibited a mitogen-dependent growth phenotype suggesting that nonautonomous SMC produce factors that actively inhibit autonomous growth. In contrast, heterokaryons constructed between Neo7 SMC and autonomous embryonic SMC retained an autonomous growth phenotype. We examined the expression of known tumor suppressors to determine if any of these factors played a role in inhibiting SMC autonomous growth. p27, p53, pRb, and PTEN were abundantly expressed by Neo7 SMC and e17 SMC under both basal and serum stimulated conditions. The data suggest that the mechanisms driving SMC replication during neointimal formation are self-driven and self-regulated, and that at specific times after injury, SMC escape normal growth suppressive mechanisms through the loss of intracellular growth suppressor activity.     

Attenuation of neointimal vascular smooth muscle cellularity in atheroma by plasminogen activator inhibitor type 1 (PAI-1).

Rupture of vulnerable atheroma often underlies acute coronary syndromes. Vulnerable plaques exhibit a paucity of vascular smooth muscle cells (VSMCs) in the cap. Therefore, decreased VSMC migration into the neointima may predispose to vulnerability. The balance between cell surface plasminogen activator activity and its inhibition [mediated primarily by plasminogen activator inhibitor type 1 (PAI-1)] modulates migration of diverse types of cells. We sought to determine whether increased expression of PAI-1 would decrease migration of VSMCs in vitro and neointimal cellularity in vivo in apolipoprotein E knockout (ApoE(-/-)) mice fed a high-fat diet. Increased vessel wall expression of PAI-1 in transgenic mice was induced with the SM22alpha promoter. VSMC migration through Matrigel in vitro was quantified with laser scanning cytometry. Expression of PAI-1 was increased threefold in the aortic wall of SM22-PAI transgene-positive mice. Neointimal cellularity of vascular lesions was decreased by 26% (p=0.01; n=5 each) in ApoE(-/-) mice with the SM22-PAI transgene compared with ApoE(-/-) mice. VSMCs explanted from transgene-positive mice exhibited twofold greater expression of PAI-1 and their migration was attenuated by 27% (p=0.03). Accordingly, increased expression of PAI-1 protein by VSMCs reduces their migration in vitro and their contribution to neointimal cellularity in vivo.     

Effect of •OH scavenging action by non-SH-containing angiotensin converting enzyme inhibitor imidaprilat using microdialysis

We examined the effect of non-SH-containing angiotensin converting enzyme (ACE) inhibitor imidaprilat on hydroxyl radical (•OH) generation using microdialysis. Salicylic acid in Ringer's solution containing sodium salicylate (0.5 n mol μL⁻¹ min⁻¹) was infused directly through a microdialysis probe to detect the generation of •OH as reflected by the formation of 2,3-dihydroxybenzoic acid (DHBA) in the myocardium of anesthetized rats. We compared the ability of two non-SH-containing ACE inhibitors (imidaprilat and enalaprilat) with an -SH-containing ACE inhibitor (captopril) to scavenge the •OH. When iron (II) was administered to animals pretreated with these three ACE inhibitors, a decrease in 2,3-DHBA of all three compounds was observed, as compared with the iron (II) only-treated group. All three ACE inhibitors were able to scavenge •OH generated by the action of iron (II). However, imidaprilat is a free radical scavenger more potent than enalaprilat. These results suggested that ACE inhibitors are probably not only related to the presence of the SH radical.     

Neointima formation after vascular injury is angiotensin II mediated.

Smooth muscle proliferation of injured blood vessels leads to pathologically significant stenosis in animals and humans. We report here the pharmacological confirmation of an involvement of angiotensin II in this process as a major, necessary mediator of neointima formation. In the rat carotid artery, an animal model of post-angioplastic restenosis, we have obtained by local intraluminal infusion of peptidic angiotensin II antagonist after balloon catheterization, suppression of neointima formation and preservation of the luminal integrity. Sham operated control animals treated without medication and operated control animals treated simultaneously with angiotensin converting enzyme inhibitor and with agonistic angiotensin II, suffered major stenosis through the myoproliferative response of the injured vessel. These results prove that angiotensin II plays a key role as a mediator of vascular neointima formation.     

Perivascular gene transfer of NADPH oxidase inhibitor suppresses angioplasty-induced neointimal proliferation of rat carotid artery

Vascular stretch induces NADPH oxidase-derived superoxide anion (O2-), which has been implicated in hypertrophy and cell proliferation. We hypothesized that targeted delivery of an NADPH oxidase inhibitor to the adventitia would reduce stretch-induced vascular O2- and attenuate neointima formation. We designed a novel replication-deficient adenovirus containing a fibroblast-active promoter driving expression of NADPH oxidase inhibitory sequence gp91ds (Ad-PDGFbetaR-gp91ds/eGFP). 1) We characterized the specificity of this promoter using pPDGFbetaR-luciferase by showing induction of luciferase in cultured rat aortic fibroblasts but not in vascular smooth muscle cells. 2) Using RT-PCR, we observed expression of gp91ds and the reporter gene in fibroblasts after infection with Ad-PDGFbetaR-gp91ds/eGFP. 3) Using Ad-CMV-eGFP as a control, we delivered Ad-PDGFbetaR-gp91ds/eGFP to the adventitia of the rat common carotid artery (CCA). Immunohistochemistry confirmed localized delivery of the inhibitor to the adventitia. After CCAs were injured with an embolectomy catheter, we observed a significant increase in neointima-to-media area ratio in control CCAs, which was significantly attenuated in CCAs treated with the gp91ds-expressing virus. In a second group of rats, we detected a 10-fold increase in distension-stimulated O2-, which was significantly reduced in CCAs infected with gp91ds-expressing virus. These data demonstrate that localized adventitial delivery of an NADPH oxidase inhibitor is effective in reducing overall vascular O2- and neointima formation, suggesting that adventitial NADPH oxidase plays a functional role in development of neointimal hyperplasia.     

Gene delivery to the vasculature mediated by low-titre adeno-associated virus serotypes 1 and 5

BACKGROUND: Vascular gene therapy requires safe and efficient gene transfer in vivo. Recombinant adeno-associated virus (AAV) is a promising viral vector but its use in the vasculature has produced conflicting results and serotypes other than AAV2 have not been intensively studied. We investigated the efficiency of alternative AAV serotypes for vascular gene delivery in vitro and in vivo. METHODS: Vascular cell lines were transduced in vitro with AAV vectors. Rabbit carotid arteries were transduced with AAV1, 2 and 5 encoding enhanced green fluorescent protein (eGFP) ( approximately 1.4 x 10(9) DNAse-resistant particles (drp)). Gene transfer in vivo was assessed at 14 and 28 days. High-titre doses of AAV2 encoding beta-galactosidase in vivo were also studied. RESULTS: In vitro, transgene expression was not observed in endothelial cells using AAV2 whereas the use of serotypes 1 and 5 resulted in detectable levels of transgene expression. Coronary artery smooth muscle cells (CASMCs) transduced with AAV2 demonstrated higher levels of GFP expression than AAV1 or 5. Transgene expression in vivo was noted using low-titre AAV1 and AAV5 ( approximately 1.4 x 10(9) drp) in the media and adventitia. Only delivery of AAV1eGFP resulted in neointimal formation (3/7 vessels examined), with transgene expression noted in the neointima. Transgene expression with AAV2 was not detected in any layer of the blood vessel wall using low titre ( approximately 10(9) drp). However, high-titre ( approximately 10(11) drp) AAV2 resulted in transduction of cells in the media and adventitia but not the endothelium. CONCLUSIONS: AAV1 and AAV5 have advantages over AAV2 for vascular gene delivery at low titres.     

As human pial arteries (internal diameter 200-1000 microm) get smaller, their wall thickness and capacity to develop tension relative to their diameter increase.

Pial arteries play a key role in the regulation of human cerebral blood flow. However, many of the features and mechanisms that regulate the tone and diameters of these vessels cannot be studied in situ. One approach is to study in vitro segments of arteries obtained during neurosurgical procedures. The ratios of arterial media thickness to lumen diameter and of the capacity to develop wall force to lumen diameter have important functional consequences and are known to change in disease. Experiments were carried out on pial arteries from normotensive humans to determine the way in which these parameters vary with vessel size. Vessel dimensions--media thickness and lumen diameter were derived from fixed sections using quantitative morphometry. Wall force was measured using a resistance artery myograph. The ratio of media thickness to lumen diameter and of maximum tension developed to lumen diameter both increased as vessel diameter decreased. These ratios do not change over the age range of 15-75 years. These findings show that although in vivo intralumenal pressure falls as human pial arteries become smaller, their media thickness and capacity to develop tone increase.