Linked mechanical and biological aspects of remodeling in mouse pulmonary arteries with hypoxia-induced hypertension

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.     

Mechanical properties of elastin along the thoracic aorta in the pig

Understanding the mechanical environment of each component within the arterial wall is fundamental for understanding vascular growth and remodelling and for engineering artificial vascular conduits. We have investigated the mechanical status of arterial elastin by measuring the circumferential mechanical properties of purified elastin as function of position along the descending thoracic aorta of the pig. The tensile circumferential secant modulus, E(sec), measured in uniaxial mechanical tests, increased 30% (P<0.001), from a value of 0.88 MPa in the proximal tissue near the aortic arch to 1.14 MPa in the distal tissue near the diaphragm, indicating the stiffness of the elastin sample increased with position. Breaking stress was 54% higher in the distal tissue compared to the proximal (P<0.001), but the breaking stretch ratio did not change. E(sec) correlated with the ratio of radius to wall thickness measured in the no load state, r(nl)/h(nl), suggesting that the rise in stiffness was linked to ring morphology. The higher stiffness and strength of the distal tissue might be explained by a higher proportion of circumferentially oriented fibres in the distal tissue, which would indicate that the elastin meshwork in the thoracic aorta may become progressively anisotropic with distance from the heart. The ratio r(nl)/(h(nl)E (sec))rose only 7%, which suggests that the in vivo circumferential strain on the elastin may be constant along the pig thoracic aorta. The positional variation in elastin's properties should be taken into account in mechanical studies on purified elastin and in mathematical models of aorta mechanics.     

Validation of a pressure diameter method for determining modulus and strain of collagen engagement for long branches of bovine pulmonary arteries

Recent studies have shown that capacitance measurements of large arteries provide better prognosis and diagnosis than tests of resistance alone in pulmonary hypertension (Mahapatra et al., 2006, "Relationship of Pulmonary Arterial Capacitance and Mortality in Idiopathic Pulmonary Arterial Hypertension," J. Am. Coll. Cardiol., 47(4), pp. 799-803; Reuben, 1971, "Compliance of the Human Pulmonary Arterial System in Disease," Circ. Res., 29, pp. 40-50]. Decreased arterial capacitance causes increased load to the heart and is the direct result of increased stiffness and elastic modulus of the arterial wall. Here, we validate a pressure-diameter (PD) method for comparing the elastic modulus and collagen engagement for post-hilar pulmonary arteries with a large range of arterial diameter. The tissue mechanics of the post-hilar arteries are not well-characterized in pulmonary hypertension. It is believed that future studies with this method will provide useful insight into the role of passive tissue mechanics of these arteries in the pathophysiology of pulmonary hypertension, eventually improving clinical diagnosis, prognosis, and treatment. Post-hilar pulmonary arteries, excised from healthy and hypertensive calves and healthy cows, were inflated over a range of 0 [mm Hg] to 110 [mm Hg] in an isolated tissue bath. Internal pressure was recorded with an electric pressure catheter. Artery diameter and longitudinal stretch were recorded photographically. Stress-strain data curves were extracted using Lame's law of thick-walled tubes. Radial strips were removed from each section and tested in a uniaxial (MTS) tester for validation. Both the elastic modulus and collagen engagement strain were similar to results obtained by more traditional means. The average difference between measured values of the two methods for collagen engagement strain was 3.3% of the average value of the engagement strain. The average difference between the measured values of the two methods for modulus of elasticity was 7.4% of the average value of the modulus. The maximum, theoretical, relative error for the stress determined with the PD method was calculated at 20.3%. The PD method proved to be a suitable replacement for uniaxial strain tests in comparing collagen engagement strains. The method allowed faster testing of tissues of multiple diameters, while removing the effect of end conditions. The PD method will be of further utility in continued study of tissue mechanics in pulmonary hypertension studies.     

Abnormal pulmonary artery stiffness in pulmonary arterial hypertension: in vivo study with intravascular ultrasound

Background

There is increasing recognition that pulmonary artery stiffness is an important determinant of right ventricular (RV) afterload in pulmonary arterial hypertension (PAH). We used intravascular ultrasound (IVUS) to evaluate the mechanical properties of the elastic pulmonary arteries (PA) in subjects with PAH, and assessed the effects of PAH-specific therapy on indices of arterial stiffness.

Method

Using IVUS and simultaneous right heart catheterisation, 20 pulmonary segments in 8 PAH subjects and 12 pulmonary segments in 8 controls were studied to determine their compliance, distensibility, elastic modulus and stiffness index β. PAH subjects underwent repeat IVUS examinations after 6-months of bosentan therapy.

Results

At baseline, PAH subjects demonstrated greater stiffness in all measured indices compared to controls: compliance (1.50±0.11×10^–2 mm^2/mmHg vs 4.49±0.43×10^–2 mm^2/mmHg, p<0.0001), distensibility (0.32±0.03%/mmHg vs 1.18±0.13%/mmHg, p<0.0001), elastic modulus (720±64 mmHg vs 198±19 mmHg, p<0.0001), and stiffness index β (15.0±1.4 vs 11.0±0.7, p = 0.046). Strong inverse exponential associations existed between mean pulmonary artery pressure and compliance (r² = 0.82, p<0.0001), and also between mean PAP and distensibility (r² = 0.79, p = 0.002). Bosentan therapy, for 6-months, was not associated with any significant changes in all indices of PA stiffness.

Conclusion

Increased stiffness occurs in the proximal elastic PA in patients with PAH and contributes to the pathogenesis RV failure. Bosentan therapy may not be effective at improving PA stiffness.     

A microstructurally driven model for pulmonary artery tissue

A new constitutive model for elastic, proximal pulmonary artery tissue is presented here, called the total crimped fiber model. This model is based on the material and microstructural properties of the two main, passive, load-bearing components of the artery wall, elastin, and collagen. Elastin matrix proteins are modeled with an orthotropic neo-Hookean material. High stretch behavior is governed by an orthotropic crimped fiber material modeled as a planar sinusoidal linear elastic beam, which represents collagen fiber deformations. Collagen-dependent artery orthotropy is defined by a structure tensor representing the effective orientation distribution of collagen fiber bundles. Therefore, every parameter of the total crimped fiber model is correlated with either a physiologic structure or geometry or is a mechanically measured material property of the composite tissue. Further, by incorporating elastin orthotropy, this model better represents the mechanics of arterial tissue deformation. These advancements result in a microstructural total crimped fiber model of pulmonary artery tissue mechanics, which demonstrates good quality of fit and flexibility for modeling varied mechanical behaviors encountered in disease states.     

Linked opening angle and histological and mechanical aspects of the proximal pulmonary arteries of healthy and pulmonary hypertensive rats and calves

Understanding how arterial remodeling changes the mechanical behavior of pulmonary arteries (PAs) is important to the evaluation of pulmonary vascular function. Early and current efforts have focused on the arteries' histological changes, their mechanical properties under in vitro mechanical testing, and their zero-stress and no-load states. However, the linkage between the histology and mechanical behavior is still not well understood. To explore this linkage, we investigated the geometry, residual stretch, and histology of proximal PAs in both adult rat and neonatal calf hypoxic models of pulmonary hypertension (PH), compared their changes due to chronic hypoxia across species, and proposed a two-layer mechanical model of artery to relate the opening angle to the stiffness ratio of the PA outer to inner layer. We found that the proximal PA remodeling in calves was quite different from that in rats. In rats, the arterial wall thickness, inner diameter, and outer layer thickness fraction all increased dramatically in PH and the opening angle decreased significantly, whereas in calves, only the arterial wall thickness increased in PH. The proposed model predicted that the stiffness ratio of the calf proximal PAs changed very little from control to hypertensive group, while the decrease of opening angle in rat proximal PAs in response to chronic hypoxia was approximately linear to the increase of the stiffness ratio. We conclude that the arterial remodeling in rat and calf proximal PAs is different and the change of opening angle can be linked to the change of the arterial histological structure and mechanics.     

Role of elastin anisotropy in structural strain energy functions of arterial tissue

The vascular wall exhibits nonlinear anisotropic mechanical properties. The identification of a strain energy function (SEF) is the preferred method to describe its complex nonlinear elastic properties. Earlier constituent-based SEF models, where elastin is modeled as an isotropic material, failed in describing accurately the tissue response to inflation–extension loading. We hypothesized that these shortcomings are partly due to unaccounted anisotropic properties of elastin. We performed inflation–extension tests on common carotid of rabbits before and after enzymatic degradation of elastin and applied constituent-based SEFs, with both an isotropic and an anisotropic elastin part, on the experimental data. We used transmission electron microscopy (TEM) and serial block-face scanning electron microscopy (SBFSEM) to provide direct structural evidence of the assumed anisotropy. In intact arteries, the SEF including anisotropic elastin with one family of fibers in the circumferential direction fitted better the inflation–extension data than the isotropic SEF. This was supported by TEM and SBFSEM imaging, which showed interlamellar elastin fibers in the circumferential direction. In elastin-degraded arteries, both SEFs succeeded equally well in predicting anisotropic wall behavior. In elastase-treated arteries fitted with the anisotropic SEF for elastin, collagen engaged later than in intact arteries. We conclude that constituent-based models with an anisotropic elastin part characterize more accurately the mechanical properties of the arterial wall when compared to models with simply an isotropic elastin. Microstructural imaging based on electron microscopy techniques provided evidence for elastin anisotropy. Finally, the model suggests a later and less abrupt collagen engagement after elastase treatment.     

Glycosaminoglycans contribute to extracellular matrix fiber recruitment and arterial wall mechanics

Elastic and collagen fibers are well known to be the major load-bearing extracellular matrix (ECM) components of the arterial wall. Studies of the structural components and mechanics of arterial ECM generally focus on elastin and collagen fibers, and glycosaminoglycans (GAGs) are often neglected. Although GAGs represent only a small component of the vessel wall ECM, they are considerably important because of their diverse functionality and their role in pathological processes. The goal of this study was to study the mechanical and structural contributions of GAGs to the arterial wall. Biaxial tensile testing was paired with multiphoton microscopic imaging of elastic and collagen fibers in order to establish the structure–function relationships of porcine thoracic aorta before and after enzymatic GAG removal. Removal of GAGs results in an earlier transition point of the nonlinear stress–strain curves \((p<0.05)\). However, stiffness was not significantly different after GAG removal treatment, indicating earlier but not absolute stiffening. Multiphoton microscopy showed that when GAGs are removed, the adventitial collagen fibers are straighter, and both elastin and collagen fibers are recruited at lower levels of strain, in agreement with the mechanical change. The amount of stress relaxation also decreased in GAG-depleted arteries \((p<0.05)\). These findings suggest that the interaction between GAGs and other ECM constituents plays an important role in the mechanics of the arterial wall, and GAGs should be considered in addition to elastic and collagen fibers when studying arterial function.     

Strain-rate sensitive mechanical properties of tendon fascicles from mice with genetically engineered alterations in collagen and decorin

Tendons have complex mechanical behaviors that are nonlinear and time dependent. It is widely held that these behaviors are provided by the tissue composition and structure. It is generally thought that type I collagen provides the primary elastic strength to tendon while proteoglycans, such as decorin, play a role in failure and viscoelastic properties. This study sought to quantify such structure-function relationships by comparing tendon mechanical properties between normal mice and mice genetically engineered for altered type I collagen content and absence of decorin. Uniaxial tensile ramp to failure experiments were performed on tail tendon fascicles at two strain rates, 0.5%/s and 50%/s. Mutations in type I collagen led to reduced failure load and stiffness with no changes in failure stress, modulus or strain rate sensitivity. Fascicles without decorin had similar elastic properties to normal fascicles, but reduced strain rate sensitivity. Fascicles from immature mice, with increased decorin content compared to adult fascicles, had inferior elastic properties but higher strain rate sensitivity. These results showed that tendon viscoelasticity is affected by decorin content but not by collagen alterations. This study provides quantitative evidence for structure-function relationships in tendon, including the role of proteoglycan in viscoelasticity.     

A Modified Verhoeff-Van Gieson Elastin Histochemical Stain to Enable Pulmonary Arterial Hypertension Model Characterization

Optimal histochemical staining is critical to ensure excellent quality stained sections to enable light microscopic and histomorphometric image analysis. Verhoeff-van Gieson is the most widely used histochemical stain for the visualization of vascular elastic fibers. However, it is notoriously difficult to differentiate fine elastic fibers of small vasculature to enable histomorphometric image analysis, especially in organs such as the lung. A tissue fixation procedure of 10% neutral buffered formalin with subsequent fixation in 70% ethanol further compounds the problem of small vessel staining and identification. Therefore, a modified Verhoeff’s elastin stain was developed as a reliable method to optimally highlight the internal and external elastic laminae of small arteries (50-100 µm external diameter) and intra-acinar vessels (10-50 µm external diameter) in 3 µm thick lung tissue sections from models of pulmonary arterial hypertension. This modified Verhoeff’s elastin stain demonstrated well-defined staining of fine elastic fibers of pulmonary blood vessels enabling subsequent histomorphometric image analysis of vessel wall thickness in small arteries and intra-acinar vessels. In conclusion, modification of the standard Verhoeff-van Gieson histochemical stain is needed to visualize small caliber vessels’ elastic fibers especially in tissues fixed in 10% neutral buffered formalin followed by additional fixation in 70% ethanol.Key words: Histochemical stain, histomorphology, lung, Verhoeff-van Gieson, elastin     

Nonlinear elastic analysis of blood vessels

Based on the theory of Green and Adkins [9], a strain energy function is proposed to describe the nonlinear mechanical behavior of arteries. The arterial tissue is assumed to be a nonlinear elastic, incompressible material with local triclinicity and transverse isotropy. Although the arterial tissue shows viscous phenomena, experimental results have indicated that viscosity is only a second-order effect as compared to the nonlinear elasticity of the tissue. The advantage of the formulation presented herein is that it is relatively simple and results in an accurate stress-strain relation that can be used readily for the study of wave propagations in the blood vessels. For nonlinear finite strain elasticity of the order two, ten elastic constants are needed to describe the material nonlinearity of the arterial tissue. Based on the orthogonal, transverse isotropies and the incompressibility conditions, ten constraint equations may be established and the elastic constants can be uniquely determined by correlating with the experimental results. An example of calculating these elastic constants is made by using the experimental data of Patel, et al. [14-17] for the intercoastal arteries in living dogs. The predicted mechanical behavior of canine arteries is quite satisfactory as compared to the experimental data except when the longitudinal and the circumferential stretches exceed 1.60. However, such a strain magnitude may not be expected in in-vivo arteries because of the constraints of peripheral connecting tissues. Otherwise, the strain energy function including higher order strain terms should be used.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in Large Pulmonary Arterial Viscoelasticity in Chronic Pulmonary Hypertension

Conduit pulmonary artery (PA) stiffening is characteristic of pulmonary arterial hypertension (PAH) and is an excellent predictor of mortality due to right ventricular (RV) overload. To better understand the impact of conduit PA stiffening on RV afterload, it is critical to examine the arterial viscoelastic properties, which require measurements of elasticity (energy storage behavior) and viscosity (energy dissipation behavior). Here we hypothesize that PAH leads to frequency-dependent changes in arterial stiffness (related to elasticity) and damping ratio (related to viscosity) in large PAs. To test our hypothesis, PAH was induced by the combination of chronic hypoxia and an antiangiogenic compound (SU5416) treatment in mice. Static and sinusoidal pressure-inflation tests were performed on isolated conduit PAs at various frequencies (0.01–20 Hz) to obtain the mechanical properties in the absence of smooth muscle contraction. Static mechanical tests showed significant stiffening of large PAs with PAH, as expected. In dynamic mechanical tests, structural stiffness (κ) increased and damping ratio (D) decreased at a physiologically relevant frequency (10 Hz) in hypertensive PAs. The dynamic elastic modulus (E), a material stiffness, did not increase significantly with PAH. All dynamic mechanical properties were strong functions of frequency. In particular, κ, E and D increased with increasing frequency in control PAs. While this behavior remained for D in hypertensive PAs, it reversed for κ and E. Since these novel dynamic mechanical property changes were found in the absence of changes in smooth muscle cell content or contraction, changes in collagen and proteoglycans and their interactions are likely critical to arterial viscoelasticity in a way that has not been previously described. The impact of these changes in PA viscoelasticity on RV afterload in PAH awaits further investigation.     

Layer-specific arterial micromechanics and microstructure: Influences of age,anatomical location,and processing technique

The importance of matrix micromechanics is increasingly recognized in cardiovascular research due to the intimate role they play in local vascular cell physiology. However, variations in micromechanics among arterial layers (i.e. intima, media, adventitia), as well as dependency on local matrix composition and/or structure, anatomical location or developmental stage remain largely unknown. This study determined layer-specific stiffness in elastic arteries, including the main pulmonary artery, ascending aorta, and carotid artery using atomic force indentation. To compare stiffness with age and frozen processing techniques, neonatal and adult pulmonary arteries were tested, while fresh (vibratomed) and frozen (cryotomed) tissues were tested from the adult aorta. Results revealed that the mean compressive modulus varied among the intima, sub-luminal media, inner-middle media, and adventitia layers in the range of 1–10 kPa for adult arteries. Adult samples, when compared to neonatal pulmonary arteries, exhibited increased stiffness in all layers except adventitia. Compared to freshly isolated samples, frozen preparation yielded small stiffness increases in each layer to varied degrees, thus inaccurately representing physiological stiffness. To interpret micromechanics measurements, composition and structure analyses of structural matrix proteins were conducted with histology and multiphoton imaging modalities including second harmonic generation and two-photon fluorescence. Composition analysis of matrix protein area density demonstrated that decrease in the elastin-to-collagen and/or glycosaminoglycan-to-collagen ratios corresponded to stiffness increases in identical layers among different types of arteries. However, composition analysis was insufficient to interpret stiffness variations between layers which had dissimilar microstructure. Detailed microstructure analyses may contribute to more complete understanding of arterial micromechanics.     

Contribution of elastin and collagen to the inflation response of the pig thoracic aorta: assessing elastin's role in mechanical homeostasis

This study was undertaken to understand elastin's role in the mechanical homeostasis of the arterial wall. The mechanical properties of elastin vary along the aorta, and we hypothesized this maintained a uniform mechanical environment for the elastin, despite regional variation in loading. Elastin's physiological loading was determined by comparing the inflation response of intact and autoclave purified elastin aortas from the proximal and distal thoracic aorta. Elastin's stretch and stress depend on collagen recruitment. Collagen recruitment started in the proximal aorta at systolic pressures (13.3 to 14.6 kPa) and in the distal at sub-diastolic pressures (9.3 to 10.6 kPa). In the proximal aorta collagen did not contribute significantly to the stress or stiffness, indicating that elastin determined the vessel properties. In the distal aorta, the circumferential incremental modulus was 70% higher than in the proximal aorta, half of which (37%) was due to a stiffening of the elastin. Compared to the elastin tissue in the proximal aorta, the distal elastin suffered higher physiological circumferential stretch (29%, P=0.03), circumferential stress (39%, P=0.02), and circumferential stiffness (37%, P=0.006). Elastin's physiological axial stresses were also higher (67%, P=0.003). These findings do not support the hypothesis that the loading on elastin is constant along the aorta as we expected from homeostasis.     

Arterial Extracellular Matrix: A Mechanobiological Study of the Contributions and Interactions of Elastin and Collagen

The complex network structure of elastin and collagen extracellular matrix (ECM) forms the primary load bearing components in the arterial wall. The structural and mechanobiological interactions between elastin and collagen are important for properly functioning arteries. Here, we examined the elastin and collagen organization, realignment, and recruitment by coupling mechanical loading and multiphoton imaging. Two-photon excitation fluorescence and second harmonic generation methods were performed with a multiphoton video-rate microscope to capture real time changes to the elastin and collagen structure during biaxial deformation. Enzymatic removal of elastin was performed to assess the structural changes of the remaining collagen structure. Quantitative analysis of the structural changes to elastin and collagen was made using a combination of two-dimensional fast Fourier transform and fractal analysis, which allows for a more complete understanding of structural changes. Our study provides new quantitative evidence, to our knowledge on the sequential engagement of different arterial ECM components in response to mechanical loading. The adventitial collagen exists as large wavy bundles of fibers that exhibit fiber engagement after 20% strain. The medial collagen is engaged throughout the stretching process, and prominent elastic fiber engagement is observed up to 20% strain after which the engagement plateaus. The fiber orientation distribution functions show remarkably different changes in the ECM structure in response to mechanical loading. The medial collagen shows an evident preferred circumferential distribution, however the fiber families of adventitial collagen are obscured by their waviness at no or low mechanical strains. Collagen fibers in both layers exhibit significant realignment in response to unequal biaxial loading. The elastic fibers are much more uniformly distributed and remained relatively unchanged due to loading. Removal of elastin produces similar structural changes in collagen as mechanical loading. Our study suggests that the elastic fibers are under tension and impart an intrinsic compressive stress on the collagen.     

Arterial Extracellular Matrix: A Mechanobiological Study of the Contributions and Interactions of Elastin and Collagen

The complex network structure of elastin and collagen extracellular matrix (ECM) forms the primary load bearing components in the arterial wall. The structural and mechanobiological interactions between elastin and collagen are important for properly functioning arteries. Here, we examined the elastin and collagen organization, realignment, and recruitment by coupling mechanical loading and multiphoton imaging. Two-photon excitation fluorescence and second harmonic generation methods were performed with a multiphoton video-rate microscope to capture real time changes to the elastin and collagen structure during biaxial deformation. Enzymatic removal of elastin was performed to assess the structural changes of the remaining collagen structure. Quantitative analysis of the structural changes to elastin and collagen was made using a combination of two-dimensional fast Fourier transform and fractal analysis, which allows for a more complete understanding of structural changes. Our study provides new quantitative evidence, to our knowledge on the sequential engagement of different arterial ECM components in response to mechanical loading. The adventitial collagen exists as large wavy bundles of fibers that exhibit fiber engagement after 20% strain. The medial collagen is engaged throughout the stretching process, and prominent elastic fiber engagement is observed up to 20% strain after which the engagement plateaus. The fiber orientation distribution functions show remarkably different changes in the ECM structure in response to mechanical loading. The medial collagen shows an evident preferred circumferential distribution, however the fiber families of adventitial collagen are obscured by their waviness at no or low mechanical strains. Collagen fibers in both layers exhibit significant realignment in response to unequal biaxial loading. The elastic fibers are much more uniformly distributed and remained relatively unchanged due to loading. Removal of elastin produces similar structural changes in collagen as mechanical loading. Our study suggests that the elastic fibers are under tension and impart an intrinsic compressive stress on the collagen.     

A Rapid Digestive Technique to Expose Networks of Vascular Elastic Fibers for Sem Observation

The NaOH sonication digestion technique permits rapid isolation and exposure of intact networks of elastic fibers in vascular tissue for 3-dimensional observation with the SEM. The configuration of the network of elastic fibers within the vascular wall of large elastic arteries (aorta) is generally agreed to be a flexible framework through which smooth muscle cells and collagenous fibers are interwoven. However, the configuration of elastic fiber networks in muscular arteries, medium sized veins and smaller vessels remains unknown. When the lengthy standard biochemical elastin purification techniques were applied to vessels containing lesser amounts of elastic tissue and finer elastic fibers, the vessels were completely digested. In contrast, the digestion and sonication technique isolated and exposed intact networks of delicate elastic fibers in blood vessels which do not contain large amounts of elastic tissue. Unfixed vessels were cut into short segments, placed in 0.5 N NaOH and sonicated for 20-40 min. The specimens were rinsed in deionized distilled H₂O, then autoclaved for 30 min. The tissue was rinsed a second time, fixed and processed routinely for SEM. Elastic stains and enzymatic digestion with chromatographically purified elastase and collagenase confirmed that the digestion and sonication technique produced clean, isolated networks of elastic fibers. Knowledge of the configuration of the networks of elastic fibers in different vessels enhances understanding of distensibility characteristics of individual vessels and serves as a baseline for studying alterations in the elastic framework which occur during aging and disease processes such as atherosclerosis, arterial hypertension and aneurysms.     

A rapid digestive technique to expose networks of vascular elastic fibers for SEM observation

The NaOH sonication digestion technique permits rapid isolation and exposure of intact networks of elastic fibers in vascular tissue for 3-dimensional observation with the SEM. The configuration of the network of elastic fibers within the vascular wall of large elastic arteries (aorta) is generally agreed to be a flexible framework through which smooth muscle cells and collagenous fibers are interwoven. However, the configuration of elastic fiber networks in muscular arteries, medium sized veins and smaller vessels remains unknown. When the lengthy standard biochemical elastin purification techniques were applied to vessels containing lesser amounts of elastic tissue and finer elastic fibers, the vessels were completely digested. In contrast, the digestion and sonication technique isolated and exposed intact networks of delicate elastic fibers in blood vessels which do not contain large amounts of elastic tissue. Unfixed vessels were cut into short segments, placed in 0.5 N NaOH and sonicated for 20-40 min. The specimens were rinsed in deionized distilled H2O, then autoclaved for 30 min. The tissue was rinsed a second time, fixed and processed routinely for SEM. Elastic stains and enzymatic digestion with chromatographically purified elastase and collagenase confirmed that the digestion and sonication technique produced clean, isolated networks of elastic fibers. Knowledge of the configuration of the networks of elastic fibers in different vessels enhances understanding of distensibility characteristics of individual vessels and serves as a baseline for studying alterations in the elastic framework which occur during aging and disease processes such as atherosclerosis, arterial hypertension and aneurysms.     

The effect of elastin damage on the mechanics of the aortic valve

Porcine bioprosthetic heart valves degenerate and fail mechanically through a mechanism that is currently not well understood. It has been suggested that damage to the elastin component of prosthetic valve cusps could be responsible for changes in the mechanical function of the valve that would predispose it to increased damage and ultimate failure. To determine whether damage to elastin can produce the structural and mechanical changes that could initiate the process of bioprosthetic valve degeneration, we developed an elastase treatment protocol that fragments elastin and negates its mechanical contribution to the valve tissue. Valve cusps were mechanically tested before and after digestion to measure the mechanical changes resulting from elastin damage. Elastin damage produced a decrease in radial and circumferential extensibility (from 43 to 18% strain radially and 12 to 7% strain circumferentially), with a slight increase in stiffness (1.3-2.6kN/m for radial and 10.6-11.9kN/m for circumferential directions). Digestions with trypsin, which does not cleave elastin, confirmed that the changes in mechanics of the circumferential samples were likely due to the nonspecific removal of proteoglycans by elastase, while the changes in the radial samples were indeed due to elastin damage. Removing the mechanical contribution of elastin alters the mechanical behavior of the aortic valve cusp, primarily in the radial direction. This finding implies that damage to elastin will distend the cusps, reduce their extensibility, and increase their stiffness. Damage to elastin may therefore contribute to the degeneration and failure of prosthetic valves.     

Suppression of tissue inhibitors of metalloproteinases may reverse severe pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a fatal disease characterized by a progressive increase in pulmonary vascular resistance and vascular remodeling leading to right heart failure and early death. The pathology of PAH is associated with endothelium dysfunction and vascular remodeling in pulmonary arteries. In diseased pulmonary arteries, the balance between matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) is broken down. In this process, TIMP are up-regulated, which inhibits MMP, promotes extracellular matrix (ECM) deposition and finally leads to vascular remodeling. So, what would happen to PAH if the expression of TIMP was down-regulated in diseased pulmonary vessels? We hypothesize that the attenuation of TIMP at the advanced stage of PAH might reverse severe PAH, via ameliorating vascular remodeling and endothelium repair.