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Elastin is a primary structural protein in the arterial wall that contributes to vascular mechanical properties and degrades with aging. Aging is associated with arterial stiffening and an increase in blood pressure. There is evidence that arterial aging follows different timelines with sex. Our objective was to investigate how elastin content affects arterial remodeling in male and female mice with aging. We used male and female wild-type ( Eln +/+ ) and elastin heterozygous ( Eln +/- ) mice at 6, 12, and 24 mo of age and measured their blood pressure and arterial morphology, wall structure, protein content, circumferential stress, stretch ratio, and stiffness. Two arteries were used with varying contents of elastin: the left common carotid and ascending aorta. We show that Eln +/- arteries start at a different homeostatic set point for circumferential wall stress, stretch, and material stiffness but show similar increases with aging to Eln +/+ mice. With aging, structural stiffness is greatly increased, while material stiffness and circumferential stress are only slightly increased, highlighting the importance of maintaining these homeostatic values. Circumferential stretch shows the smallest change with age and may be important for controlling cellular phenotype. Independent sex differences are mostly associated with males being larger than females; however, many of the measured factors show age × sex and/or genotype × sex interactions, indicating that males and females follow different cardiovascular remodeling timelines with aging and are differentially affected by reduced elastin content. NEW & NOTEWORTHY A comprehensive study on arterial mechanical behavior as a function of elastin content, aging, and sex in mice. Elastin haploinsufficient arteries start at a different homeostatic set point for mechanical parameters such as circumferential stress, stretch, and material stiffness. Structural stiffness of the arterial wall greatly increases with aging, as expected, but there are interactions between sex and aging for most of the mechanical parameters that are important to consider in future work.

Objective: Elastin gene deletion or mutation leads to arterial stenoses due to vascular smooth muscle cell (SMC) proliferation. Human induced pluripotent stem cells-derived SMCs can model the elastin insufficiency phenotype in vitro but show only partial rescue with rapamycin. Our objective was to identify drug candidates with superior efficacy in rescuing the SMC phenotype in elastin insufficiency patients. Approach and Results: SMCs generated from induced pluripotent stem cells from 5 elastin insufficiency patients with severe recurrent vascular stenoses (3 Williams syndrome and 2 elastin mutations) were phenotypically immature, hyperproliferative, poorly responsive to endothelin, and exerted reduced tension in 3-dimensional smooth muscle biowires. Elastin mRNA and protein were reduced in SMCs from patients compared to healthy control SMCs. Fourteen drug candidates were tested on patient SMCs. Of the mammalian target of rapamycin inhibitors studied, everolimus restored differentiation, rescued proliferation, and improved endothelin-induced calcium flux in all patient SMCs except one Williams syndrome. Of the calcium channel blockers, verapamil increased SMC differentiation and reduced proliferation in Williams syndrome patient cells but not in elastin mutation patients and had no effect on endothelin response. Combination treatment with everolimus and verapamil was not superior to everolimus alone. Other drug candidates had limited efficacy., Conclusions: Everolimus caused the most consistent improvement in SMC differentiation, proliferation and in SMC function in patients with both syndromic and nonsyndromic elastin insufficiency, and offers the best candidate for drug repurposing for treatment of elastin insufficiency associated vasculopathy.

Deposition of elastin and collagen in the aorta correlates with increases in blood pressure and flow during development, suggesting that the aorta adjusts its mechanical properties in response to hemodynamic stresses. Elastin knockout (Eln -/- ) mice have high blood pressure and pathological remodeling of the aorta and die soon after birth. We hypothesized that decreasing blood pressure in Eln -/- mice during development may reduce hemodynamic stresses and alleviate pathological remodeling of the aorta. We treated Eln +/+ and Eln -/- mice with the anti-hypertensive medication captopril throughout embryonic development and then evaluated left ventricular (LV) pressure and aortic remodeling at birth. We found that captopril treatment decreased Eln -/- LV pressure to values near Eln +/+ mice and alleviated the wall thickening and changes in mechanical behavior observed in untreated Eln -/- aorta. The changes in thickness and mechanical behavior in captopril-treated Eln -/- aorta were not due to alterations in measured elastin or collagen amounts, but may have been caused by alterations in smooth muscle cell (SMC) properties. We used a constitutive model to understand how changes in stress contributions of each wall component could explain the observed changes in composite mechanical behavior. Our modeling results show that alterations in the collagen natural configuration and SMC properties in the absence of elastin may explain untreated Eln -/- aortic behavior and that partial rescue of the SMC properties may account for captopril-treated Eln -/- aortic behavior.

Rationale: Elastin is an important ECM (extracellular matrix) protein in large and small arteries. Vascular smooth muscle cells (SMCs) produce the layered elastic laminae found in elastic arteries but synthesize little elastin in muscular arteries. However, muscular arteries have a well-defined internal elastic lamina (IEL) that separates endothelial cells (ECs) from SMCs. The extent to which ECs contribute elastin to the IEL is unknown., Objective: To use targeted elastin (Eln) deletion in mice to explore the relative contributions of SMCs and ECs to elastic laminae formation in different arteries., Methods and Results: We used SMC- and EC-specific Cre recombinase transgenes with a novel floxed Eln allele to focus gene inactivation in mice. Inactivation of Eln in SMCs using Sm22aCre resulted in depletion of elastic laminae in the arterial wall with the exception of the IEL and SMC clusters in the outer media near the adventitia. Inactivation of elastin in ECs using Tie2Cre or Cdh5Cre resulted in normal medial elastin and a typical IEL in elastic arteries. In contrast, the IEL was absent or severely disrupted in muscular arteries. Interruptions in the IEL resulted in neointimal formation in the ascending aorta but not in muscular arteries., Conclusions: Combined with lineage-specific fate mapping systems, our knockout results document an unexpected heterogeneity in vascular cells that produce the elastic laminae. SMCs and ECs can independently form an IEL in most elastic arteries, whereas ECs are the major source of elastin for the IEL in muscular and resistance arteries. Neointimal formation at IEL disruptions in the ascending aorta confirms that the IEL is a critical physical barrier between SMCs and ECs in the large elastic arteries. Our studies provide new information about how SMCs and ECs contribute elastin to the arterial wall and how local elastic laminae defects may contribute to cardiovascular disease.

Purpose: Lax eyelid syndrome (LES) is defined as the association of distensible "floppy" eyelids and chronic papillary conjunctivitis. LES is also found in patients with obstructive sleep apnea (OSA) who have systemic elevation of inflammatory markers, including matrix metalloproteinases (MMP). Locally elevated MMP levels have also been demonstrated co-localized with elastin loss in eyelids of patients with LES, accounting for their "floppiness." The purpose of this study was to investigate tear film MMP levels and determine their association with eyelid laxity and OSA. We also evaluated 3 previous grading systems to determine the severity of lid laxity and introduced a new "laxometer" device., Methods: Thirty-seven subjects underwent bilateral eyelid laxity assessments prior to polysomnography testing. OSA severity was graded using the apnea hypopnea index (AHI). The degree of eyelid laxity was determined using three published methods and a newly proposed "laxometer" method. Commercially available InflammaDry ® kits were used to determine the presence of MMP-9 in the tear film., Results: There was a significant elevation in tear MMP-9 levels in patients with LES compared to controls (p < .05). Of the 37 total patients enrolled in this study, 2 patients (5.4%) did not have sleep study results. Thirty-two of the remaining 35 patients (91.4%) were determined to have OSA (AHI > 5). In this sample, there was no meaningful association between OSA and MMP-9 (p = .12). Although there were positive associations between OSA severity, laxometer measurements, and previously established grading methods, none achieved statistical significance (all p > .05)., Conclusions: There was an elevation of MMP-9 in tears of patients with LES. Elevated tear MMP-9 was also not associated with OSA. Although there is some evidence to support the association of eyelid laxity and OSA, the most accurate and reliable method for grading eyelid laxity remains unclear., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Elastin deficiency causes vascular stiffening, a leading risk for hypertension and chronic kidney disease (CKD). The mechanisms mediating hypertension and/or CKD pathogenesis due to elastin deficiency are poorly understood. Using the elastin heterozygous (Eln+/-) mouse model, we tested whether renal dysfunction due to elastin deficiency occurs independently of and precedes the development of hypertension. We assessed blood pressure and renal hemodynamics in 30-day and 12-week-old male and female mice. At P30, blood pressure of Eln+/- mice was similar to wild-type controls; however, renal blood flow was lower, whereas renal vascular resistance was augmented at baseline in Eln+/- mice. At 12 weeks, renal vascular resistance remained elevated while filtration fraction was higher in male Eln+/- relative to wild-type mice. Heterozygous mice showed isolated systolic hypertension that was evident only at nighttime. Acute salt loading with 6% dietary sodium increased daytime systolic blood pressure only in male Eln+/- mice, causing a rightward shift and blunted slope of the pressure-natriuresis curve. Renal interlobar artery basal tone and myogenic response to increasing intraluminal pressure at day 10 were similar, whereas they were augmented at day 30 and at 12 weeks old in Eln+/- mice, and normalized by the AT1R blocker, candesartan. Heterozygous mice also exhibited podocyte foot process damage that persisted even when blood pressure was normalized to wild-type levels with hydralazine. Thus, elastin insufficiency triggers structural defects and abnormal remodeling of renal vascular signaling involving AT1R-mediated vascular mechanotransduction and renal hyperfiltration with increased blood pressure sensitivity to dietary sodium contributing to systolic hypertension., (Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Objective: Elastin deficiency because of heterozygous loss of an ELN allele in Williams syndrome causes obstructive aortopathy characterized by medial thickening and fibrosis and consequent aortic stiffening. Previous work in Eln -null mice with a severe arterial phenotype showed that inhibition of mTOR (mechanistic target of rapamycin), a key regulator of cell growth, lessened the aortic obstruction but did not prevent early postnatal death. We investigated the effects of mTOR inhibition in Eln -null mice partially rescued by human ELN that manifest a less severe arterial phenotype and survive long term., Approach and Results: Thoracic aortas of neonatal and juvenile mice with graded elastin deficiency exhibited increased signaling through both mTOR complex 1 and 2. Despite lower predicted wall stress, there was increased phosphorylation of focal adhesion kinase, suggestive of greater integrin activation, and increased transforming growth factor-β-signaling mediators, associated with increased collagen expression. Pharmacological blockade of mTOR by rapalogs did not improve luminal stenosis but reduced mechanosignaling (in delayed fashion after mTOR complex 1 inhibition), medial collagen accumulation, and stiffening of the aorta. Rapalog administration also retarded somatic growth, however, and precipitated neonatal deaths. Complementary, less-toxic strategies to inhibit mTOR via altered growth factor and nutrient responses were not effective., Conclusions: In addition to previously demonstrated therapeutic benefits of rapalogs decreasing smooth muscle cell proliferation in the absence of elastin, we find that rapalogs also prevent aortic fibrosis and stiffening attributable to partial elastin deficiency. Our findings suggest that mTOR-sensitive perturbation of smooth muscle cell mechanosensing contributes to elastin aortopathy., (© 2017 American Heart Association, Inc.)

Objective: Williams syndrome is characterized by obstructive aortopathy attributable to heterozygous loss of ELN , the gene encoding elastin. Lesions are thought to result primarily from excessive smooth muscle cell (SMC) proliferation and consequent medial expansion, although an initially smaller caliber and increased stiffness of the aorta may contribute to luminal narrowing. The relative contributions of such abnormalities to the obstructive phenotype had not been defined., Approach and Results: We quantified determinants of luminal stenosis in thoracic aortas of Eln -/- mice incompletely rescued by human ELN . Moderate obstruction was largely because of deficient circumferential growth, most prominently of ascending segments, despite increased axial growth. Medial thickening was evident in these smaller diameter elastin-deficient aortas, with medial area similar to that of larger diameter control aortas. There was no difference in cross-sectional SMC number between mutant and wild-type genotypes at multiple stages of postnatal development. Decreased elastin content was associated with medial fibrosis and reduced aortic distensibility because of increased structural stiffness but preserved material stiffness. Elastin-deficient SMCs exhibited greater contractile-to-proliferative phenotypic modulation in vitro than in vivo. We confirmed increased medial collagen without evidence of increased medial area or SMC number in a small ascending aorta with thickened media of a Williams syndrome subject., Conclusions: Deficient circumferential growth is the predominant mechanism for moderate obstructive aortic disease resulting from partial elastin deficiency. Our findings suggest that diverse aortic manifestations in Williams syndrome result from graded elastin content, and SMC hyperplasia causing medial expansion requires additional elastin loss superimposed on ELN haploinsufficiency., (© 2017 American Heart Association, Inc.)

Increased arterial stiffness is a common characteristic of humans with Williams-Beuren syndrome and mouse models of elastin insufficiency. Arterial stiffness is associated with multiple negative cardiovascular outcomes, including myocardial infarction, stroke, and sudden death. Therefore, identifying therapeutic interventions that improve arterial stiffness in response to changes in elastin levels is of vital importance. The goal of this study was to determine the effect of chronic pharmacologic therapy with different classes of antihypertensive medications on arterial stiffness in elastin insufficiency. Elastin-insufficient mice 4-6 wk of age and wild-type littermates were subcutaneously implanted with osmotic micropumps delivering a continuous dose of one of the following: vehicle, losartan, nicardipine, or propranolol for 8 wk. At the end of treatment period, arterial blood pressure and large artery compliance and remodeling were assessed. Our results show that losartan and nicardipine treatment lowered blood pressure and pulse pressure in elastin-insufficient mice. Elastin and collagen content of abdominal aortas as well as ascending aorta and carotid artery biomechanics were not affected by any of the drug treatments in either genotype. By reducing pulse pressure and shifting the working pressure range of an artery to a more compliant region of the pressure-diameter curve, antihypertensive medications may mitigate the consequences of arterial stiffness, an effect that is drug class independent. These data emphasize the importance of early recognition and long-term management of hypertension in Williams-Beuren syndrome and elastin insufficiency., (Copyright © 2015 the American Physiological Society.)

Aortopathy is characterized by vascular smooth muscle cell (VSMC) abnormalities and elastic fiber fragmentation. Elastin insufficient (Eln (+/-)) mice demonstrate latent aortopathy similar to human disease. We hypothesized that aortopathy manifests primarily in the aorto-pulmonary septal (APS) side of the thoracic aorta due to asymmetric cardiac neural crest (CNC) distribution. Anatomic (aortic root vs. ascending aorta) and molecular (APS vs. non-APS) regions of proximal aorta tissue were examined in adult and aged wild type (WT) and mutant (Eln (+/-)) mice. CNC, VSMCs, elastic fiber architecture, proteoglycan expression, morphometrics and biomechanical properties were examined using histology, 3D reconstruction, micropipette aspiration and in vivo magnetic resonance imaging (MRI). In the APS side of Eln (+/-) aorta, Sonic Hedgehog (SHH) is decreased while SM22 is increased. Elastic fiber architecture abnormalities are present in the Eln (+/-) aortic root and APS ascending aorta, and biglycan is increased in the aortic root while aggrecan is increased in the APS aorta. The Eln (+/-) ascending aorta is stiffer than the aortic root, the APS side is thicker and stiffer than the non-APS side, and significant differences in the individual aortic root sinuses are observed. Asymmetric structure-function abnormalities implicate regional CNC dysregulation in the development and progression of aortopathy.

Background: Ascending aortic dissection and rupture remain a life-threatening complication in patients with Marfan syndrome. The extracellular matrix provides strength and elastic recoil to the aortic wall, thereby preventing radial expansion. We have previously shown that ascending aortic aneurysm formation in Marfan mice (Fbn1(C1039G/+)) is associated with decreased aortic wall elastogenesis and increased elastin breakdown. In this study, we test the feasibility of quantifying aortic wall elastin content using MRI with a gadolinium-based elastin-specific magnetic resonance contrast agent in Fbn1(C1039G/+) mice., Methods and Results: Ascending aorta elastin content was measured in 32-week-old Fbn1(C1039G/+) mice and wild-type (n=9 and n=10, respectively) using 7-T MRI with a T1 mapping sequence. Significantly lower enhancement (ie, lower R1 values, where R1=1/T1) was detected post-elastin-specific magnetic resonance contrast agent in Fbn1(C1039G/+) compared with wild-type ascending aortas (1.15±0.07 versus 1.36±0.05; P<0.05). Post-elastin-specific magnetic resonance contrast agent R1 values correlated with ascending aortic wall gadolinium content directly measured by inductively coupled mass spectroscopy (P=0.006)., Conclusions: Herein, we demonstrate that MRI with elastin-specific magnetic resonance contrast agent accurately measures elastin bound gadolinium within the aortic wall and detects a decrease in aortic wall elastin in Marfan mice compared with wild-type controls. This approach has translational potential for noninvasively assessing aneurysm tissue changes and risk, as well as monitoring elastin content in response to therapeutic interventions., (© 2014 American Heart Association, Inc.)

Elastin (Eln) insufficiency in mice and humans is associated with hypertension and altered structure and mechanical properties of large arteries. However, it is not known to what extent functional or structural changes in resistance arteries contribute to the elevated blood pressure that is characteristic of Eln insufficiency. Here, we investigated how Eln insufficiency affects the structure and function of the resistance vasculature. A functional profile of resistance vasculature in Eln(+/-) mice was generated by assessing small mesenteric artery (MA) contractile and vasodilatory responses to vasoactive agents. We found that Eln haploinsufficiency had a modest effect on phenylephrine-induced vasoconstriction, whereas ANG II-evoked vasoconstriction was markedly increased. Blockade of ANG II type 2 receptors with PD-123319 or modulation of Rho kinase activity with the inhibitor Y-27632 attenuated the augmented vasoconstriction, whereas acute Y-27632 administration normalized blood pressure in Eln(+/-) mice. Sodium nitroprusside- and isoproterenol-induced vasodilatation were normal, whereas ACh-induced vasodilatation was severely impaired in Eln(+/-) MAs. Histologically, the number of smooth muscle layers did not change in Eln(+/-) MAs; however, an additional discontinuous layer of Eln appeared between the smooth muscle layers that was absent in wild-type arteries. We conclude that high blood pressure arising from Eln insufficiency is due partly to permanent changes in vascular tone as a result of increased sensitivity of the resistance vasculature to circulating ANG II and to impaired vasodilatory mechanisms arising from endothelial dysfunction characterized by impaired endothelium-dependent vasodilatation. Eln insufficiency causes augmented ANG II-induced vasoconstriction in part through a novel mechanism that facilitates contraction evoked by ANG II type 2 receptors and altered G protein signaling.

Objective: Patients with elastin deficiency attributable to gene mutation (supravalvular aortic stenosis) or chromosomal microdeletion (Williams syndrome) are characterized by obstructive arteriopathy resulting from excessive smooth muscle cell (SMC) proliferation, mural expansion, and inadequate vessel size. We investigated whether rapamycin, an inhibitor of the cell growth regulator mammalian target of rapamycin (mTOR) and effective against other SMC proliferative disorders, is of therapeutic benefit in experimental models of elastin deficiency., Approach and Results: As previously reported, Eln(-/-) mice demonstrated SMC hyperplasia and severe stenosis of the aorta, whereas Eln(+/-) mice exhibited a smaller diameter aorta with more numerous but thinner elastic lamellae. Increased mTOR signaling was detected in elastin-deficient aortas of newborn pups that was inhibited by maternal administration of rapamycin. mTOR inhibition reduced SMC proliferation and aortic obstruction in Eln(-/-) pups and prevented medial hyperlamellation in Eln(+/-) weanlings without compromising aortic size. However, rapamycin did not prolong the survival of Eln(-/-) pups, and it retarded the somatic growth of juvenile Eln(+/-) and Eln(+/+) mice. In cell cultures, rapamycin inhibited prolonged mTOR activation and enhanced proliferation of SMC derived from patients with supravalvular aortic stenosis and with Williams syndrome., Conclusions: mTOR inhibition may represent a pharmacological strategy to treat diffuse arteriopathy resulting from elastin deficiency.

Objective: • To examine functional and molecular changes of the bladders from elastin-haploinsufficient mice (Eln(+/-) ) at baseline as well as in response to partial bladder outlet obstruction (pBOO)., Materials and Methods: • Female Eln(+/-) and wild type (Wt) mice (3-4 months old) were studied. • The bladder elastin content was quantified by measuring desmosine. • Mice were divided into two groups to undergo surgery to create pBOO or to undergo sham surgery. Three days after surgery, bladder function was evaluated by in vivo cystometry, and the contractile response of bladder strips exposed to electrical field stimulation (EFS) and carbachol was examined by ex vivo myography., Results: • The Eln(+/-) -sham mice had a 33.6% decrease in bladder elastin compared with Wt-sham mice. • Cystometry showed significantly decreased bladder compliance and capacity in Eln(+/-) -sham vs Wt-sham mice; pBOO increased bladder compliance and capacity to a greater extent in Eln(+/-) mice compared with Wt mice. • Bladder strips from Eln(+/-) -sham mice showed a significantly heightened contractile response to both EFS and carbachol compared with Wt-sham mice. • A significantly increased contractile response to carbachol was detected in Wt-pBOO vs Wt-sham but not between Eln(+/-) -pBOO and Eln(+/-) -sham mice., Conclusion: • The results that elastin-deficient mice had decreased bladder compliance and capacity and increased bladder contractility; and that Wt-pBOO mice showed an enhanced contractile response to carbachol, but Eln(+/-) -pBOO mice did not, suggest that elastin is critical for normal bladder function and is involved in bladder response to pBOO., (© 2011 BJU INTERNATIONAL.)

Objective: The goal of this study was to determine whether antagonizing microRNA (miR)-29 enhances elastin (ELN) levels in cells and tissues lacking ELN., Methods and Results: miR-29 mimics reduced ELN levels in fibroblasts and smooth muscle cells, whereas miR-29 inhibition increased ELN levels. Antagonism of miR-29 also increased ELN levels in cells from patients haploinsufficient for ELN and in bioengineered human vessels., Conclusion: miR-29 antagonism may promote increased ELN levels during conditions of ELN deficiencies.

A hallmark feature of Williams-Beuren Syndrome (WBS) is a generalized arteriopathy due to elastin deficiency, presenting as stenoses of medium and large arteries and leading to hypertension and other cardiovascular complications. Deletion of a functional NCF1 gene copy has been shown to protect a proportion of WBS patients against hypertension, likely through reduced NADPH-oxidase (NOX)-mediated oxidative stress. DD mice, carrying a 0.67 Mb heterozygous deletion including the Eln gene, presented with a generalized arteriopathy, hypertension, and cardiac hypertrophy, associated with elevated angiotensin II (angII), oxidative stress parameters, and Ncf1 expression. Genetic (by crossing with Ncf1 mutant) and/or pharmacological (with ang II type 1 receptor blocker, losartan, or NOX inhibitor apocynin) reduction of NOX activity controlled hormonal and biochemical parameters in DD mice, resulting in normalized blood pressure and improved cardiovascular histology. We provide strong evidence for implication of the redox system in the pathophysiology of the cardiovascular disease in a mouse model of WBS. The phenotype of these mice can be ameliorated by either genetic or pharmacological intervention reducing NOX activity, likely through reduced angII-mediated oxidative stress. Therefore, anti-NOX therapy merits evaluation to prevent the potentially serious cardiovascular complications of WBS, as well as in other cardiovascular disorders mediated by similar pathogenic mechanism., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Elastin fibers confer passive recoil to many tissues including the lung, skin, and arteries. In the penis, elastin is present in sinusoids, arterioles, and in the tunica albuginea. Although decreased penile elastin has been reported in men with erectile dysfunction, the exact role of elastin in physiologic processes integral to erection remains speculative., Aim: The aim of this study was to characterize erectile function in elastin-deficient mice., Methods: Elastin haploinsufficient mice (Eln(+/-) ) and aged match Eln(+/+) (Wt) mice were used. Cavernosum was removed from some mice for quantification of elastin, collagen, and smooth muscle actin. Ex vivo assessment of contractile force generation was performed by myography. In vivo assessment of intracorporal pressure normalized to mean arterial pressure in response to electrical stimulation of the cavernosal nerve was measured. Veno-occlusive function was determined by cavernosography., Main Outcome Measures: The main outcome measures of this study were the in vitro and in vivo assessment of cavernosal vasoreactivity, veno-occlusive function and erection in mice deficient in elastin., Results: Eln (+/-) mice exhibited ∼33% less penile elastin than Wt mice, with no change in collagen. Cavernosal tissue from Eln(+/-) mice has a significantly heightened contractile response, explained in part by increased smooth muscle cell content. Veno-occlusive function was significantly altered in Eln(+/-) mice. Interestingly, erectile function was impaired only at submaximal voltage (1 V) stimulation (there was no impairment during the higher 2-V stimulus)., Conclusions: Eln (+/-) mice display a cavernosal phenotype consistent with developmental changes attributable to the loss of elastin. These alterations confer a degree of altered erectile function that is able to be overridden by maximal stimulatory input. Altogether, these data suggest that elastin is important for erectile function., (© 2011 International Society for Sexual Medicine.)

Pyrroline-5-carboxylate reductase 1 (PYCR1) catalyzes the last step in proline synthesis. Deficiency of PYCR1, caused by a defect in PYCR1, was recently described in patients with cutis laxa, intrauterine growth retardation, developmental dysplasia of the hips and mental retardation. In this paper, we describe additional six patients (ages ranging from 4 months to 55 years) from four Iranian families with clinical manifestations of a wrinkly skin disorder. All patients have distinct facial features comprising triangular face, loss of adipose tissue and thin pointed nose. Additional features are short stature, wrinkling over dorsum of hand and feet, visible veins over the chest and hyperextensible joints. Three of the patients from a large consanguineous family do not have mental retardation, while the remaining three patients from three unrelated families have mental and developmental delay. Mutation analysis revealed the presence of disease-causing variants in PYCR1, including a novel deletion of the entire PYCR1 gene in one family, and in each of the other patients the homozygous missense mutations c.616G > A (p.Gly206Arg), c.89T > A (p.Ile30Lys) and c.572G > A (p.Gly191Glu) respectively, the latter two of which are novel. Light- and electron microscopy investigations of skin biopsies showed smaller and fragmented elastic fibres, abnormal morphology of the mitochondria and their cristae, and slightly abnormal collagen fibril diameters with irregular outline and variable size. In conclusion, this study adds information on the natural course of PYCR1 deficiency and sheds light on the pathophysiology of this disorder. However, the exact pathogenesis of this new disorder and the role of proline in the development of the clinical phenotype remain to be fully explained.

Rationale: Elastin is a ubiquitous extracellular matrix protein that is highly organized in heart valves and arteries. Because elastic fiber abnormalities are a central feature of degenerative valve disease, we hypothesized that elastin-insufficient mice would manifest viable heart valve disease., Objective: To analyze valve structure and function in elastin-insufficient mice (Eln(+/-)) at neonatal, juvenile, adult, and aged adult stages., Methods and Results: At birth, histochemical analysis demonstrated normal extracellular matrix organization in contrast to the aorta. However, at juvenile and adult stages, thin elongated valves with extracellular matrix disorganization, including elastin fragment infiltration of the annulus, were observed. The valve phenotype worsened by the aged adult stage with overgrowth and proteoglycan replacement of the valve annulus. The progressive nature of elastin insufficiency was also shown by aortic mechanical testing that demonstrated incrementally abnormal tensile stiffness from juvenile to adult stages. Eln(+/-) mice demonstrated increased valve interstitial cell proliferation at the neonatal stage and varied valve interstitial cell activation at early and late stages. Gene expression profile analysis identified decreased transforming growth factor-beta-mediated fibrogenesis signaling in Eln(+/-) valve tissue. Juvenile Eln(+/-) mice demonstrated normal valve function, but progressive valve disease (predominantly aortic regurgitation) was identified in 17% of adult and 70% of aged adult Eln(+/-) mice by echocardiography., Conclusions: These results identify the Eln(+/-) mouse as a model of latent aortic valve disease and establish a role for elastin dysregulation in valve pathogenesis.

Elastin is an essential component of vertebrate arteries that provides elasticity and stores energy during the cardiac cycle. Elastin production in the arterial wall begins midgestation but increases rapidly during the last third of human and mouse development, just as blood pressure and cardiac output increase sharply. The aim of this study is to characterize the structure, hemodynamics, and mechanics of developing arteries with reduced elastin levels and determine the critical time period where elastin is required in the vertebrate cardiovascular system. Mice that lack elastin (Eln(-/-)) or have approximately one-half the normal level (Eln(+/-)) show relatively normal cardiovascular development up to embryonic day (E) 18 as assessed by arterial morphology, left ventricular blood pressure, and cardiac function. Previous work showed that just a few days later, at birth, Eln(-/-) mice die with high blood pressure and tortuous, stenotic arteries. During this period from E18 to birth, Eln(+/-) mice add extra layers of smooth muscle cells to the vessel wall and have a mean blood pressure 25% higher than wild-type animals. These findings demonstrate that elastin is only necessary for normal cardiovascular structure and function in mice starting in the last few days of fetal development. The large increases in blood pressure during this period may push hemodynamic forces over a critical threshold where elastin becomes required for cardiovascular function. Understanding the interplay between elastin amounts and hemodynamic forces in developing vessels will help design treatments for human elastinopathies and optimize protocols for tissue engineering.

Williams-Beuren syndrome (WBS) is caused by a submicroscopic deletion on chromosome 7q11.23 that encompasses the entire elastin (ELN) gene. Elastin, a key component of elastic fibers within the lung, is progressively destroyed in emphysema. Defects in the elastin gene have been associated with increased susceptibility towards developing chronic obstructive pulmonary disease (COPD) and emphysema in both humans and mice. We postulate that hemizygosity at the elastin gene locus may increase susceptibility towards the development of COPD and emphysema in subjects with WBS. We describe an adult subject with WBS who was a lifelong non-smoker and was found to have moderate emphysema. We also examined the pulmonary function of a separate cohort of adolescents and young adults with WBS. Although no significant spirometric abnormalities were identified, a significant proportion of subjects reported respiratory symptoms. Thus, while significant obstructive disease does not appear to be common in relatively young adults with WBS, subclinical emphysema and lung disease may exist which possibly could worsen with advancing age. Further investigation may elucidate the pathogenesis of non-smoking-related emphysema., ((c) 2010 Wiley-Liss, Inc.)

Objective: Even though elastin and fibrillin-1 are the major structural components of elastic fibers, mutations in elastin and fibrillin-1 lead to narrowing of large arteries in supravalvular aortic stenosis and dilation of the ascending aorta in Marfan syndrome, respectively. A genetic approach was therefore used here to distinguish the differential contributions of elastin and fibrillin-1 to arterial development and compliance., Methods and Results: Key parameters of cardiovascular function were compared among adult mice haploinsufficient for elastin (Eln(+/-)), fibrillin-1 (Fbn1(+/-)), or both proteins (dHet). Physiological and morphological comparisons correlate elastin haploinsufficiency with increased blood pressure and vessel length and tortuosity in dHet mice, and fibrillin-1 haploinsufficiency with increased aortic diameter in the same mutant animals. Mechanical tests confirm that elastin and fibrillin-1 impart elastic recoil and tensile strength to the aortic wall, respectively. Additional ex vivo analyses demonstrate additive and overlapping contributions of elastin and fibrillin-1 to the material properties of vascular tissues. Lastly, light and electron microscopy evidence implicates fibrillin-1 in the hypertension-promoted remodeling of the elastin-deficient aorta., Conclusions: These results demonstrate that elastin and fibrillin-1 have both differential and complementary roles in arterial wall formation and function, and advance our knowledge of the structural determinants of vascular physiology and disease.

Elastin is a major structural component of large elastic arteries and a principal determinant of arterial biomechanical properties. Elastin loss-of-function mutations in humans have been linked to the autosomal-dominant disease supravalvular aortic stenosis, which is characterized by stenotic lesions in both the systemic and pulmonary circulations. To better understand how elastin insufficiency influences the pulmonary circulation, we evaluated pulmonary cardiovascular physiology in a unique set of transgenic and knockout mice with graded vascular elastin dosage (range 45-120% of wild type). The central pulmonary arteries of elastin-insufficient mice had smaller internal diameters (P < 0.0001), thinner walls (P = 0.002), and increased opening angles (P = 0.002) compared with wild-type controls. Pulmonary circulatory pressures, measured by right ventricular catheterization, were significantly elevated in elastin-insufficient mice (P < 0.0001) and showed an inverse correlation with elastin level. Although elastin-insufficient animals exhibited mild to moderate right ventricular hypertrophy (P = 0.0001) and intrapulmonary vascular remodeling, the changes were less than expected, given the high right ventricular pressures, and were attenuated compared with those seen in hypoxia-induced models of pulmonary arterial hypertension. The absence of extensive pathological cardiac remodeling at the high pressures in these animals suggests a developmental adaptation designed to maintain right-sided cardiac output in a vascular system with altered elastin content.

Background: Genital prolapse is a debilitating manifestation of pelvic floor dysfunction. The cause of this condition has not been elucidated. The purpose of this study was to determine elastin content and RNA expression of related enzymes of elastin synthesis in uterosacral ligament biopsies from women with severe prolapse, and controls with normal pelvic support., Methods: Biopsies were taken from the uterosacral ligament tissue of 31 women with Grade III or greater prolapse and 29 women with normal pelvic support. Elastin content was assessed by measuring desmosine using radioimmunoassay, and quantitative real time PCR was performed to quantify mRNA levels of lysyl oxidase (LOX), lysyl oxidase like-1 (LOXL1), LOXL2 and fibulin-5 (FIB-5)., Results: The mean desmosine concentration found in uterosacral ligaments of women with prolapse (n =26) was 103.3+/-59.3 pmolD/mgP compared to controls (n =29) 120.5+/-47.4 pmolD/mgP (p =0.1943). In the subgroup of subjects with complete procidentia (n =8), mean desmosine concentration was 50.6+/-25.8 and 127.1+/-42.2 pmolD/mgP in age-matched controls (n =12) (p <0.05). In tissue from subjects with more than 2 vaginal deliveries (n =18), the mean desmosine concentration was 99.9+/-60.7 and 133.0+/-44.0 pmolD/mgP in controls (n =17) (p <0.05). Expression of LOX, LOXL1 and LOXL2 decreased 8.2-fold+/-3.4, 5.0-fold+/-1.7 and 15.2-fold+/-5.2, respectively (mean+/-SD) in cases versus controls (p<0.05). Expression of FIB-5 was increased 3.1-fold+/-0.7 compared to controls (p<0.05)., Conclusions: Significantly decreased desmosine content was measured in the uterosacral ligament tissue from women with prolapse versus controls in women with parity >2 and in women with complete procidentia. Suppression of mRNA for LOX and two LOX isoenzymes was correspondingly present. These results suggest that altered elastin metabolism is present in women with uterine prolapse.

Elastic fibers provide recoil to tissues that undergo repeated stretch, such as the large arteries and lung. These large extracellular matrix (ECM) structures contain numerous components, and our understanding of elastic fiber assembly is changing as we learn more about the various molecules associated with the assembly process. The main components of elastic fibers are elastin and microfibrils. Elastin makes up the bulk of the mature fiber and is encoded by a single gene. Microfibrils consist mainly of fibrillin, but also contain or associate with proteins such as microfibril associated glycoproteins (MAGPs), fibulins, and EMILIN-1. Microfibrils were thought to facilitate alignment of elastin monomers prior to cross-linking by lysyl oxidase (LOX). We now know that their role, as well as the overall assembly process, is more complex. Elastic fiber formation involves elaborate spatial and temporal regulation of all of the involved proteins and is difficult to recapitulate in adult tissues. This report summarizes the known interactions between elastin and the microfibrillar proteins and their role in elastic fiber assembly based on in vitro studies and evidence from knockout mice. We also propose a model of elastic fiber assembly based on the current data that incorporates interactions between elastin, LOXs, fibulins and the microfibril, as well as the pivotal role played by cells in structuring the final functional fiber., (Copyright 2008 Wiley-Liss, Inc.)

Rationale: Lung hypoplasia in congenital diaphragmatic hernia (CDH) seems to involve impaired alveolar septation. We hypothesized that disturbed deposition of elastin and expression of fibroblast growth factor 18 (FGF18), an elastogenesis stimulus, occurs in CDH., Objectives: To document FGF18 and elastin in human CDH and ovine surgical and rat nitrofen models and to use models to evaluate the benefit of treatments., Methods: Human CDH and control lungs were collected post mortem. Diaphragmatic hernia was created in sheep at 85 days; fetal lungs were collected at 139 days (term = 145 days). Pregnant rats received nitrofen at 12 days; fetal lungs were collected at 21 days (term = 22 days). Some of the sheep fetuses with hernia underwent tracheal occlusion (TO); some of the nitrofen-treated pregnant rats received vitamin A. Both treatments are known to promote lung growth., Measurements and Main Results: Coincidental with the onset of secondary septation, FGF18 protein increased threefold in control human lungs, which failed to occur in CDH. FGF18 labeling was found in interstitial cells of septa. Elastin staining demonstrated poor septation and markedly decreased elastin density in CDH lungs. Consistently, lung FGF18 transcripts were diminished 60 and 83% by CDH in sheep and rats, respectively, and elastin density and expression were diminished. TO and vitamin A restored FGF18 and elastin expression in sheep and rats, respectively. TO restored elastin density., Conclusions: Impaired septation in CDH is associated with decreased FGF18 expression and elastic fiber deposition. Simultaneous correction of FGF18 and elastin defects by TO and vitamin A suggests that defective elastogenesis may result, at least partly, from FGF18 deficiency.

Elastin is an extracellular matrix protein that is the primary component of elastic fibers, and is expressed in the great vessels as well as the semilunar and atrioventricular valves. Elastin haploinsufficiency, resulting from mutation or deletion of the elastin gene, is an important clinical problem that is typically characterized by arteriopathy. Herein is described a patient with elastin haploinsufficiency due to partial deletion of the Williams-Beuren syndrome region, resulting in bilateral semilunar valve disease and arteriopathy. Histochemical analysis of the aortic valve revealed decreased and disorganized elastin with loss of the normal trilaminar cusp organization. These findings suggest that elastin has a role in the pathogenesis of semilunar valve disease.

Supravalvular aortic stenosis (SVAS) is associated with decreased elastin and altered arterial mechanics. Mice with a single deletion in the elastin gene (ELN(+/-)) are models for SVAS. Previous studies have shown that elastin haploinsufficiency in these mice causes hypertension, decreased arterial compliance, and changes in arterial wall structure. Despite these differences, ELN(+/-) mice have a normal life span, suggesting that the arteries remodel and adapt to the decreased amount of elastin. To test this hypothesis, we performed in vitro mechanical tests on abdominal aorta, ascending aorta, and left common carotid artery from ELN(+/-) and wild-type (C57BL/6J) mice. We compared the circumferential and longitudinal stress-stretch relationships and residual strains. The circumferential stress-stretch relationship is similar between genotypes and changes <3% with longitudinal stretch at lengths within 10% of the in vivo value. At mean arterial pressure, the circumferential stress in the ascending aorta is higher in ELN(+/-) than in wild type. Although arterial pressures are higher, the increased number of elastic lamellae in ELN(+/-) arteries results in similar tension/lamellae compared with wild type. The longitudinal stress-stretch relationship is similar between genotypes for most arteries. Compared with wild type, the in vivo longitudinal stretch is lower in ELN(+/-) abdominal and carotid arteries and the circumferential residual strain is higher in ELN(+/-) ascending aorta. The increased circumferential residual strain brings the transmural strain distribution in ELN(+/-) ascending aorta close to wild-type values. The mechanical behavior of ELN(+/-) arteries is likely due to the reduced elastin content combined with adaptive remodeling during vascular development.

Background: In 1968, De Barsy reported on a girl exhibiting an aged aspect, 'dwarfism, oligophrenia, and degeneration of the elastic tissue in cornea and skin'. The disorder was recognized as a subgroup of cutis laxa syndrome and termed De Barsy-Moens-Dierckx syndrome. The pathogenesis of the disorder is unknown., Methods: To improve the comprehension of the pathogenetic mechanisms involved in the De Barsy syndrome, we performed an ultrastructural, morphometric, immunocytochemical study on a skin biopsy of a boy with the De Barsy phenotype, who has been clinically followed for 12 years from birth. Moreover, the lysyl oxidase activity was measured on skin fibroblasts cultured in vitro., Results: Light and electron microscopy, morphometry, and immunocytochemical observations showed a significant reduction of the elastic fibers in the papillary and in the reticular dermis of patient compared to an age-matched control (p < 0.05). By contrast, the collagen structure, content, and the distribution were normal, as well as lysyl oxidase activity in the medium of in vitro fibroblasts (12,323 DPM/10(6) cells). The immunoreaction for antibodies recognizing fibrillin-1, neutrophilic elastase, and tumor necrosis factor-alpha was stronger, whereas that for antibodies against transforming growth factor-beta was less pronounced in the dermis of the De Barsy boy compared to control., Conclusions: Clinical, phenotypic, and structural data were consistent with the diagnosis of De Barsy syndrome. This is the first case described in Italy. Clinical and structural data confirm that the elastic component is mostly affected in this disorder. Moreover, ultrastructural and immunochemical findings suggest that both elastic fiber degradative and very likely synthetic processes are involved.

Cardiovascular disease is largely a consequence of coronary artery blockage through excessive proliferation of smooth muscle cells. It in turn leads to myocardial infarction and permanent and functionally devastating tissue damage to the heart wall. Our studies have revealed that elastin is a primary player in maintaining vascular smooth muscle cells in their dormant state and thus may be a useful therapeutic in vascular disease. By studying zebrafish, which unlike humans, can repair damage to heart muscle, we have begun to uncover some of the genes that seem necessary to undertake the de-differentiation steps that currently fail and prevent the formation of new proliferating cardiomyocytes at the site of damage in a mammalian heart.

Mice haploinsufficient for elastin develop structural changes in vessel walls similar to those seen in patients with mutations in the elastin gene. A new study demonstrates that due to mechanical changes in the vessel wall, these animals exhibit increased mean arterial pressures. The results evoke the possibility that alterations in elastin may contribute to the development of essential hypertension in patients.

Supravalvular aortic stenosis is an autosomal-dominant disease of elastin (Eln) insufficiency caused by loss-of-function mutations or gene deletion. Recently, we have modeled this disease in mice (Eln+/-) and found that Eln haploinsufficiency results in unexpected changes in cardiovascular hemodynamics and arterial wall structure. Eln+/- animals were found to be stably hypertensive from birth, with a mean arterial pressure 25-30 mmHg higher than their wild-type counterparts. The animals have only moderate cardiac hypertrophy and live a normal life span with no overt signs of degenerative vascular disease. Examination of arterial mechanical properties showed that the inner diameters of Eln+/- arteries were generally smaller than wild-type arteries at any given intravascular pressure. Because the Eln+/- mouse is hypertensive, however, the effective arterial working diameter is comparable to that of the normotensive wild-type animal. Physiological studies indicate a role for the renin-angiotensin system in maintaining the hypertensive state. The association of hypertension with elastin haploinsufficiency in humans and mice strongly suggests that elastin and other proteins of the elastic fiber should be considered as causal genes for essential hypertension.

Williams syndrome (WS) is due to a deletion in the WS critical region at 7q11.23 which includes the elastin gene (ELN). One of the most characteristic features of this disorder is a harsh, brassy, or hoarse voice but the etiology of the vocal characteristics are unknown. We report two patients with WS who had bilateral vocal cord abnormalities, bringing to four the number of children with WS in whom such defects have been documented. We suggest that vocal cord abnormalities may be a far more common feature of WS than has been previously suspected, and that mild vocal cord dysfunction caused by abnormal vocal cord elastin may be the cause of the hoarse voice in this condition., (Copyright 2003 Wiley-Liss, Inc.)

To elucidate the pathomechanism leading to obstructive vascular disease in patients with elastin deficiency, we compared both elastogenesis and proliferation rate of cultured aortic smooth-muscle cells (SMCs) and skin fibroblasts from five healthy control subjects, four patients with isolated supravalvular aortic stenosis (SVAS), and five patients with Williams-Beuren syndrome (WBS). Mutations were determined in each patient with SVAS and in each patient with WBS. Three mutations found in patients with SVAS were shown to result in null alleles. RNA blot hybridization, immunostaining, and metabolic labeling experiments demonstrated that SVAS cells and WBS cells have reduced elastin mRNA levels and that they consequently deposit low amounts of insoluble elastin. Although SVAS cells laid down approximately 50% of the elastin made by normal cells, WBS cells deposited only 15% of the elastin made by normal cells. The observed difference in elastin-gene expression was not caused by a difference in the stability of elastin mRNA in SVAS cells compared with WBS cells, but it did indicate that gene-interaction effects may contribute to the complex phenotype observed in patients with WBS. Abnormally low levels of elastin deposition in SVAS cells and in WBS cells were found to coincide with an increase in proliferation rate, which could be reversed by addition of exogenous insoluble elastin. We conclude that insoluble elastin is an important regulator of cellular proliferation. Thus, the reduced net deposition of insoluble elastin in arterial walls of patients with either SVAS or WBS leads to the increased proliferation of arterial SMCs. This results in the formation of multilayer thickening of the tunica media of large arteries and, consequently, in the development of hyperplastic intimal lesions leading to segmental arterial occlusion.

Hurler disease resulting from a deficiency in alpha-L-iduronidase, which causes an accumulation of dermatan sulfate and heparan sulfate glycosaminoglycans, is characterized by connective tissue and skeletal deformations, cardiomyopathy, cardiac valve defects, and progressive coronary artery stenosis. In this report, we present evidence that accumulation of dermatan sulfate but not heparan sulfate moieties is linked to impaired elastic fiber assembly that, in turn, contributes substantially to the development of the clinical phenotype in Hurler disease. Our data suggest that dermatan sulfate-bearing moieties bind to and cause functional inactivation of the 67-kd elastin-binding protein, a molecular chaperone for tropoelastin, which normally facilitates its secretion and assembly into elastic fibers. We demonstrate that, in contrast to normal skin fibroblasts and cells from Sanfilippo disease, which accumulate heparan sulfate, Hurler fibroblasts show reduced expression of elastin-binding protein and do not assemble elastic fibers, despite an adequate synthesis of tropoelastin and sufficient production of a microfibrillar scaffold of elastic fibers. Because cultured Hurler fibroblasts proliferate more quickly than their normal counterparts and the addition of exogenous insoluble elastin reduces their proliferation, we suggest that cell contacts with insoluble elastin play an important role in controlling their proliferation.

The elastin gene is consistently deleted in Williams syndrome and as this protein represents the major component of the elastic fibers of the dermis, we sought to investigate skin elastic fibers in Williams syndrome as a key to unraveling extracellular matrix disorganization in this condition. Both morphometric parameters analyzed by using automated image analysis and immunofluorescence labeling with monoclonal antibodies against elastin and fibrillin 1 showed a disorganized pre-elastic (oxytalan and elaunin) and mature elastic fibers in the dermis of 10 Williams syndrome patients compared with five healthy children and one patient with isolated supravalvular aortic stenosis. Skin biopsies in Williams syndrome patients provide a simple mean to elucidate extracellular matrix anomalies. Hopefully, this method could give clues to the understanding of the elastic network anomalies in this condition and even to the consequences of these latter on elasticity and resilience of other tissues such as the arterial tree., (Copyright 1999 Wiley-Liss, Inc.)

Background: Loss of elastin in the aortic wall is an early event in abdominal aortic aneurysm (AAA). An imbalance in the protease-antiprotease system is proposed to be one of the factors that promote connective tissue destruction. We hypothesize that plasma from AAA patients will have a reduced inhibitory capacity in comparison to normal controls., Materials and Methods: Using an assay we developed, plasma (10 microliters), collected from AAA patients (n = 14) and normal controls (n = 13), was added to the elastase inhibition assay containing succinylated elastin substrate. The reaction was initiated with 13.9 units porcine pancreatic elastase (PPE). Elastase activity in the presence and absence of plasma was compared. Plasma elastase was also determined using the Merck PMN-elastase kit., Results: The relative activity of exogenous elastase (%) in the presence of AAA plasma (n = 14, mean age 73.4 years +/- 1.7 SEM) was 42.59% +/- 4.3 SEM, while that in the presence of control plasma (n = 13, mean age 73.9 years +/- 2.1 SEM) was 10.23% +/- 2.1 SEM (P < 0.0001). Analysis of plasma elastase (microgram/L) indicated that there was no significant difference between normal (n = 9, 207.33 microgram/L +/- 58.67 SEM) and AAA (n = 9, 145.34 microgram/L +/- 29.54 SEM) (P = 0.359)., Conclusion: There is a significant reduction in the plasma inhibitory capacity of elastase in AAA patients in comparison to normal controls, though plasma elastase level was not significantly different. The data presented here give experimental evidence to the protease-antiprotease imbalance in AAA patient plasma and may lead to the development of a measurable parameter to monitor AAA., (Copyright 1999 Academic Press.)

Elastin, the main component of the extracellular matrix of arteries, was thought to have a purely structural role. Disruption of elastin was believed to lead to dissection of arteries, but we showed that mutations in one allele encoding elastin cause a human disease in which arteries are blocked, namely, supravalvular aortic stenosis. Here we define the role of elastin in arterial development and disease by generating mice that lack elastin. These mice die of an obstructive arterial disease, which results from subendothelial cell proliferation and reorganization of smooth muscle. These cellular changes are similar to those seen in atherosclerosis. However, lack of elastin is not associated with endothelial damage, thrombosis or inflammation, which occur in models of atherosclerosis. Haemodynamic stress is not associated with arterial obstruction in these mice either, as the disease still occurred in arteries that were isolated in organ culture and therefore not subject to haemodynamic stress. Disruption of elastin is enough to induce subendothelial proliferation of smooth muscle and may contribute to obstructive arterial disease. Thus, elastin has an unanticipated regulatory function during arterial development, controlling proliferation of smooth muscle and stabilizing arterial structure.

In recent years a rising interest has developed among the authors studying the problems of pathogenesis and treatment of chronic bronchitis and pulmonary emphysema in the factors responsible for the immune equilibrium of the respiratory system. In particular, the correlation has been stressed between the levels of inflammation mediators and the activity of proteolytic enzymes and their inhibitors in lung tissue. Most studies seem to confirm the validity of the elastase-antielastase concept. Among the environmental factors directly responsible for the development of these diseases tobacco smoke and other irritant fumes (SO2NO2) are mentioned. This concept had been the foundation for trials of therapeutic use of inhibitors of elastolytic enzymes.