Elastic Lamellae Research Articles

Propensity to calcification as a pathway to renal hypoxia in chronic kidney disease and in hypertension.

Propensity to calcification as a pathway to renal hypoxia in chronic kidney disease and in hypertension.

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta.

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic lamellae and smooth muscle cells (SMCs), and an outer layer of fibroblasts and extracellular matrix. In contrast to the widespread study of pathological models (e.g., atherosclerosis) in the adult aorta, much less is known about the embryonic and perinatal aorta. Here, we focus on SMCs and provide protocols for the analysis of the morphogenesis and pathogenesis of embryonic and perinatal aortic SMCs in normal development and disease. Specifically, the four protocols included are: i) in vivo embryonic fate mapping and clonal analysis; ii) explant embryonic aorta culture; iii) SMC isolation from the perinatal aorta; and iv) subcutaneous osmotic mini-pump placement in pregnant (or non-pregnant) mice. Thus, these approaches facilitate the investigation of the origin(s), fate, and clonal architecture of SMCs in the aorta in vivo. They allow for modulating embryonic aorta morphogenesis in utero by continuous exposure to pharmacological agents. In addition, isolated aortic tissue explants or aortic SMCs can be used to gain insights into the role of specific gene targets during fundamental processes such as muscularization, proliferation, and migration. These hypothesis-generating experiments on isolated SMCs and the explanted aorta can then be assessed in the in vivo context through pharmacological and genetic approaches.

Abstract P511: Targeting Il-1β Protects From Aortic Aneurysms Induced by Disrupted Tgfβ Signaling in Smooth Muscle Cells

Aortic aneurysms represent a life-threatening condition because of the current lack of effective treatments. Aneurysm formation is typically associated with extracellular matrix remodeling and persistent inflammation. Although the molecular mechanisms underlying aortic pathology remain largely unclear, TGFβ signaling is unquestionably implied and its downstream target Smad4 showed protective functions for maintenance of aortic walls’ integrity. Using mice with smooth muscle cells (SMCs) specific deletion of Smad4 in the adult ( Smad4 -SMC iko ), developing spontaneous aneurysms (Ascending Aorta Diameter: Smad4 -SMC iko 2.15±0.03; Smad4 -SMC wt 1.7±0.03;***p< 0.001), we investigated the molecular mechanisms activated by dysregulation of TGFβ signaling. Structural disarrangement of ascending aorta in Smad4 -SMC iko mice was clearly appreciated early after Smad4 deletion as discrete breaks of elastic lamellae (breaks/section: Smad4 -SMC iko 2.05±0.5; Smad4 -SMC wt 0.83±0.4;***p< 0.001). Interestingly, the islands of damage in the aorta of Smad4 -SMC iko were enriched of immune infiltrate, mainly monocytes/macrophages, as indicated by FACS and immunofluorescence. We then analyzed several pathways downstream to Smad4 inhibition, finding a selective activation of NF-kB/IL-1β in SMCs. To test the relevance of this pathway in the formation of aneurysms, we deleted Smad4 in SMCs of mice with Il1r1 null background ( Smad4 -SMC iko ; Il1r1 -/- ). Ultrasonographic analyses revealed that ablation of IL1 receptor1 protected Smad4 -SMC iko mice from the progression of pathology and improved their overall survival. In the end, to test the translational potential of our findings, we neutralized IL-1β signaling with the clinically relevant murine version of the FDA-approved clinical drug canakinumab. During a time course of 16 weeks, while a weekly administration of control immunoglobulins did not change aneurysm progression in Smad4 -SMC iko mice, treatment with anti-IL-1β antibody significantly hampered aneurysm formation in the aorta ( Smad4 -SMC iko +anti- IL-1β 1.85±0.02; Smad4 -SMC iko +anti-IgG 2.09±0.03; ***p< 0.001) These findings identify a mechanistic target for controlling aneurysms progression induced by disrupted TGFβ signaling.

Dysregulation of Calpain Proteolytic Systems Underlies Degenerative Vascular Disorders.

Chronic vascular diseases such as atherosclerosis, aneurysms, diabetic angiopathy/retinopathy as well as fibrotic and proliferative vascular diseases are generally complicated by the progression of degenerative insults, which are characterized by endothelial dysfunction, apoptotic/necrotic cell death in vascular/immune cells, remodeling of extracellular matrix or breakdown of elastic lamella. Increasing evidence suggests that dysfunctional calpain proteolytic systems and defective calpain protein metabolism in blood vessels contribute to degenerative disorders. In vascular endothelial cells, the overactivation of conventional calpains consisting of calpain-1 and -2 isozymes can lead to the disorganization of cell-cell junctions, dysfunction of nitric oxide synthase, sensitization of Janus kinase/signal transducer and activator of transcription cascades and depletion of prostaglandin I2, which contributes to degenerative disorders. In addition to endothelial cell dysfunctions, calpain overactivation results in inflammatory insults in macrophages and excessive fibrogenic/proliferative signaling in vascular smooth muscle cells. Moreover, calpain-6, a non-proteolytic unconventional calpain, is involved in the conversion of macrophages to a pro-atherogenic phenotype, leading to the pinocytotic deposition of low-density lipoprotein cholesterol in the cells. Here, we discuss the recent progress that has been made in our understanding of how calpain contributes to degenerative vascular disorders.

Curcumin attenuates the development of thoracic aortic aneurysm by inhibiting VEGF expression and inflammation.

Angiogenesis is an important process in the pathogenesis of aortic aneurysm. The aim of the present study was to investigate the angiogenic balance and the expression of vascular endothelial growth factor (VEGF) in thoracic aortic aneurysm (TAA). A previous study demonstrated that curcumin exerts a marked effect on aortic aneurysm development. Therefore, the present study determined whether curcumin is able to modulate angiogenesis and inflammatory signaling in TAA by collecting human TAA samples and establishing a rat TAA model using periaortic application of CaCl2. TAA rats were treated with curcumin or 1% carboxymethyl cellulose and were sacrificed 4 weeks after the operation. All tissue specimens were analyzed by histological staining, immunohistochemistry and western blotting. Human TAA samples exhibited increased neovascularization and VEGF expression when compared with normal aortic walls. In rat tissues, treatment with curcumin resulted in reduced aneurysm size and restored the wavy structure of the elastic lamellae. In addition, curcumin decreased neovascularization and the expression of VEGF. Immunohistochemical analysis indicated that curcumin significantly inhibited infiltration of cluster of differentiation (CD)3+ and CD68+ cells in TAA. Furthermore, curcumin treatment decreased the expression of vascular cell adhesion molecule-1, intracellular adhesion molecule-1, monocyte chemoattractant protein-1 and tumor necrosis factor-α. Collectively, the results demonstrated that angiogenesis and VEGF expression were increased in the aortic wall in TAA. Treatment with curcumin inhibited TAA development in rats, which was associated with suppression of VEGF expression. In addition, curcumin attenuated inflammatory cell infiltration and suppressed inflammatory factor expression in the periaortic tissue of TAA.

Sex, pregnancy and aortic disease in Marfan syndrome.

BackgroundSex-related differences as well as the adverse effect of pregnancy on aortic disease outcome are well-established phenomena in humans with Marfan syndrome (MFS). The underlying mechanisms of these observations are largely unknown.ObjectivesIn an initial (pilot) step we aimed to confirm the differences between male and female MFS patients as well as between females with and without previous pregnancy. We then sought to evaluate whether these findings are recapitulated in a pre-clinical model and performed in-depth cardiovascular phenotyping of mutant male and both nulliparous and multiparous female Marfan mice. The effect of 17β-estradiol on fibrillin-1 protein synthesis was compared in vitro using human aortic smooth muscle cells and fibroblasts.ResultsOur small retrospective study of aortic dimensions in a cohort of 10 men and 20 women with MFS (10 pregnant and 10 non-pregnant) confirmed that aortic root growth was significantly increased in the pregnant group compared to the non-pregnant group (0.64mm/year vs. 0.12mm/year, p = 0.018). Male MFS patients had significantly larger aortic root diameters compared to the non-pregnant and pregnant females at baseline and follow-up (p = 0.002 and p = 0.007, respectively), but no significant increase in aortic root growth was observed compared to the females after follow-up (p = 0.559 and p = 0.352). In the GT-8/+ MFS mouse model, multiparous female Marfan mice showed increased aortic diameters when compared to nulliparous females. Aortic dilatation in multiparous females was comparable to Marfan male mice. Moreover, increased aortic diameters were associated with more severe fragmentation of the elastic lamellae. In addition, 17β-estradiol was found to promote fibrillin-1 production by human aortic smooth muscle cells.ConclusionsPregnancy-related changes influence aortic disease severity in otherwise protected female MFS mice and patients. There may be a role for estrogen in the female sex protective effect.

UCP-2 is involved in angiotensin-II-induced abdominal aortic aneurysm in apolipoprotein E-knockout mice.

UCP-2 shows an important role in modulating of mitochondrial membrane potential and cell apoptosis. Whether or not UCP-2 could been a critical factor in preventing AAA formation is not known. We report that UCP-2 protein and mRNA expression were significantly higher in Ang-Ⅱ-induced AAA of mice. The incident rate of AAA in UCP-2-/-ApoE-/- mice after Ang-Ⅱtreatment was higher than the rate in the UCP-2+/+ApoE-/- mice. The abdominal aorta from UCP-2-/-ApoE-/- mice showed the medial hypertrophy, fragmentation of elastic lamellas and depletion of α-SMA. The NADPH oxidase activity and level of MDA was significantly higher in UCP-2-/-ApoE-/- mice than UCP-2+/+ApoE-/- or WT mice. Besides, the SOD activity is increased in UCP-2+/+ApoE-/- mice as compared with WT mice, whereas deficiency of UCP-2 decreased the increasing SOD activity in Ang-Ⅱ treated ApoE-/- mice. UCP-2 knockout up-regulated the MMP2 and MMP9 expression in aortic aneurysm. Ang-Ⅱ induced apoptosis of VSMCs was increased in UCP-2-/-ApoE-/- mice. And the expression of eNOS in vascular tissue from UCP-2-/-ApoE-/- mice is lower than WT and UCP-2+/+ApoE-/- mice. This study provides a mechanism by which UCP-2, via anti-oxidants and anti-apoptosis, participates in the preventing of AAA formation.

Loss of Smooth Muscle α-Actin Leads to NF-κB-Dependent Increased Sensitivity to Angiotensin II in Smooth Muscle Cells and Aortic Enlargement.

Mutations in ACTA2, encoding the smooth muscle isoform of α-actin, cause thoracic aortic aneurysms, acute aortic dissections, and occlusive vascular diseases. We sought to identify the mechanism by which loss of smooth muscle α-actin causes aortic disease. Acta2-/- mice have an increased number of elastic lamellae in the ascending aorta and progressive aortic root dilation as assessed by echocardiography that can be attenuated by treatment with losartan, an angiotensin II (AngII) type 1 receptor blocker. AngII levels are not increased in Acta2-/- aortas or kidneys. Aortic tissue and explanted smooth muscle cells from Acta2-/- aortas show increased production of reactive oxygen species and increased basal nuclear factor κB signaling, leading to an increase in the expression of the AngII receptor type I a and activation of signaling at 100-fold lower levels of AngII in the mutant compared with wild-type cells. Furthermore, disruption of smooth muscle α-actin filaments in wild-type smooth muscle cells by various mechanisms activates nuclear factor κB signaling and increases expression of AngII receptor type I a. These findings reveal that disruption of smooth muscle α-actin filaments in smooth muscle cells increases reactive oxygen species levels, activates nuclear factor κB signaling, and increases AngII receptor type I a expression, thus potentiating AngII signaling in vascular smooth muscle cells without an increase in the exogenous levels of AngII.

Abstract 108: Deletion of Lrp1 In SMCS Differentially Alters Susceptibility of Distinct Vascular Beds to BAPN-Induced Aneurysm and Dissection Formation

Lysyl oxidase (LOX) inhibition by β-aminopropionitrile (BAPN) induces aortic aneurysm and dissection (AD). The LDL receptor-related protein 1 (LRP1) is an endocytic receptor that plays a key role in maintaining the structural integrity of vessels. Deletion of LRP1 in vascular smooth muscle cells (smLRP1 -/-) in mice induces fully penetrant and spontaneous aneurysms with aging. The objective of this study was to investigate the role of smLRP1 in modulating the susceptibility of various vascular beds to BAPN-induced AD. Wild-type (WT) and smLRP1 -/- mice were treated with BAPN supplemented in drinking water (3g/L). After 4-16 weeks, the vessels were analyzed histologically and with micro-CT. Abnormal elastic lamellae were identified throughout the vasculature of both the BAPN-treated WT and smLRP1 -/- cohorts. smLRP1 -/- mice were more susceptible to aneurysm formation after 8 (p=0.035) or 16 weeks (p=0.043) of treatment. Aneurysms in BAPN-treated WT mice were localized to the aorta, while aneurysms in BAPN-treated smLRP1 -/- mice were localized to the visceral and iliac arteries (Table). Histologic and radiographic evidence of AD was detected in the ascending and descending thoracic aorta in BAPN and AngII (subcutaneous,1μg/kg/min, 24 hrs)-treated WT mice, but not in BAPN and Ang II-treated smLRP1 -/- mice (Figure). Thoracic ruptures were more prevalent in the BAPN-treated WT group and abdominal ruptures were more prevalent in the BAPN-treated smLRP1 -/-group (p=0.02). In summary, these data demonstrate that genetic deletion of LRP1 in SMCs differentially alters the susceptibility of distinct vascular beds to the development of aneurysm and dissection upon LOX inhibition.

Abstract 300: Targeting IL-1β Protects From Aortic Aneurysms Induced by Disrupted TGFβ signaling in SMCs

Aortic aneurysms represent a life-threatening condition because of the current lack of effective treatment, with therapeutic options limited to emergency surgery. Aneurysm formation is typically associated with extracellular matrix remodeling and persistent inflammation. Although the molecular mechanisms underlying aortic pathology remain largely unclear, TGFβ signaling is unquestionably implied and its downstream target Smad4 showed protective functions for maintenance of aortic walls’ integrity. Using mice with smooth muscle cells (SMCs) specific deletion of Smad4 in the adult ( Smad4 -SMC iko ), developing spontaneous aneurysms, we investigated the molecular mechanisms activated by dysregulation of TGFβ signaling. Structural disarrangement of ascending aorta media in Smad4 -SMC iko mice was clearly appreciated early after Smad4 deletion as discrete breaks of elastic lamellae. Interestingly, the islands of damage evidenced in the aorta of Smad4 -SMC iko were enriched of immune infiltrate, mainly composed by monocytes/macrophages, as indicated by flow cytometry and immunofluorescence. We then analyzed several pathways downstream to Smad4 inhibition, finding a selective activation of NF-kB/IL-1β pathway in SMCs. This danger signal released by SMCs later recruits innate immunity, hence arising inflammation. In order to test the relevance of this pathway in the formation of aneurysms induced by TGFβ dysregulation, we deleted Smad4 in SMCs of mice with Il1r1 null background ( Smad4 -SMC iko ; Il1r1 -/- ). Serial ultrasonographic analyses revealed that ablation of IL1 receptor 1 protected mice with the SMCs deletion of Smad4 from the progression of pathology and improved their overall survival. In the end, to test the translational potential of our findings, we neutralized IL-1β signaling with the clinically relevant murine version of the FDA-approved clinical drug canakinumab. During a time course of 16 weeks, while a weekly administration of control immunoglobulins did not change aneurysm progression in Smad4 -SMC iko mice, treatment with anti-IL-1β antibody significantly hampered aneurysm formation in the aorta. These findings identify a mechanistic target for controlling aneurysms progression induced by disrupted TGFβ signaling.

Inhibition or deletion of angiotensin II type 1 receptor suppresses elastase-induced experimental abdominal aortic aneurysms

ObjectiveAngiotensin (Ang) II type 1 receptor (AT1) activation is essential for the development of exogenous Ang II-induced abdominal aortic aneurysms (AAAs) in hyperlipidemic animals. Experimental data derived from this modeling system, however, provide limited insight into the role of endogenous Ang II in aneurysm pathogenesis. Consequently, the potential translational value of AT1 inhibition in clinical AAA disease management remains incompletely understood on the basis of the existing literature. MethodsAAAs were created in wild-type (WT) and AT1a knockout (KO) mice by intra-aortic infusion of porcine pancreatic elastase (PPE). WT mice were treated with the AT1 receptor antagonist telmisartan, 10 mg/kg/d in chow, or the peroxisome proliferator-activated receptor γ (PPARγ) antagonist GW9662, 3 mg/kg/d through oral gavage, beginning 1 week before or 3 days after PPE infusion. Influences on aneurysm progression as well as mechanistic insights into AT1-mediated pathogenic processes were determined using noninvasive ultrasound imaging, histopathology, aortic gene expression profiling, and flow cytometric analysis. ResultsAfter PPE infusion, aortic enlargement was almost completely abrogated in AT1a KO mice compared with WT mice. As defined by a ≥50% increase in aortic diameter, no PPE-infused, AT1a KO mouse actually developed an AAA. On histologic evaluation, medial smooth muscle cellularity and elastic lamellae were preserved in AT1a KO mice compared with WT mice, with marked attenuation of mural angiogenesis and leukocyte infiltration. In WT mice, telmisartan administration effectively suppressed aneurysm pathogenesis after PPE infusion as well, regardless of whether treatment was initiated before or after aneurysm creation or continued for a limited or extended time. Telmisartan treatment was associated with reduced messenger RNA levels for CCL5 and matrix metalloproteinases 2 and 9 in aneurysmal aortae, with no apparent effect on PPARγ-regulated gene expression. Administration of the PPARγ antagonist GW9662 failed to “rescue” the aneurysm phenotype in telmisartan-treated, PPE-infused WT mice. Neither effector T-cell differentiation nor regulatory T-cell cellularity was affected by telmisartan treatment status. ConclusionsTelmisartan effectively suppresses the progression of elastase-induced AAAs without apparent effect on PPARγ activation or T-cell differentiation. These findings reinforce the critical importance of endogenous AT1 activation in experimental AAA pathogenesis and reinforce the translational potential of AT1 inhibition in medical aneurysm disease management.

Blockade of HMGB1 preserves vascular homeostasis and improves blood perfusion in rats of acute limb ischemia/reperfusion

Acute limb ischemia is one of the most common peripheral arterial disease, while surgical restoration of blood flow often results in ischemia/reperfusion (I/R) injury. Our previous study revealed the inflammation intensity in arterial tissue, characterized by expression of high mobility group box protein 1 (HMGB1), was contrary to the fluctuation of hemodynamics in reperfusion limbs in a rat model. This study meant to clarify the role of HMGB1 during this process. Laser Doppler perfusion imaging evaluated limb hemodynamics in mean and max perfusion unit (PU). Femoral arterial tissue was collected for molecular biology examination. The results revealed that HMGB1 promoted vascular structure remodeling and vasomotor dysfunction during acute I/R, characterized by degradation of collagenous fibers, disruption of elastic lamellae, intensive inflammation and phenotype transfer of smooth muscle cells. Blockade of HMGB1 preserved vascular homeostasis and improved PUs (PmeanPU<0.001, PmaxPU<0.001). The elevated expression of TNF-α, IL-6, ICAM, VCAM, MMP-2, MMP-9, α-SM actin correlated with HMGB1 positively. In conclusion, HMGB1 promoted vascular remodeling and dysfunction via initiating an inflammation cascade during I/R. Blockade of HMGB1 would preserve vascular homeostasis and facilitate the blood perfusion of ischemic limb.

Routine argyrophil techniques detect Rickettsia rickettsii in tissues of patients with fatal Rocky Mountain spotted fever

Rickettsia rickettsii, a bacterial tickborne pathogen that causes Rocky Mountain spotted fever (RMSF), stains poorly or not at all with conventional tissue Gram techniques, and contemporary visualization of the pathogen in formalin-fixed, paraffin-embedded tissues has relied almost entirely on immunohistochemical staining methods that are generally limited to specialized research laboratories or national reference centers. To our knowledge, previously described argyrophil-based histochemical techniques have not successfully detected rickettsiae in formalin-fixed, paraffin-embedded tissues. To investigate the ability of standard silver impregnation techniques to demonstrate the occurrence and distribution of R. rickettsii in tissues of patients with RMSF confirmed by molecular and immunohistochemical methods, three widely recognized and commercially available silver impregnation methods (Warthin–Starry, Steiner, and Dieterle’s) were applied to various tissues obtained at autopsy from 10 patients with fatal RMSF. R. rickettsii bacteria were demonstrated in one or more tissues of all patients, using each of the argyrophil-based methods, and appeared as small, dark brown-to-black lanceolate rods, often in pairs and occasionally surrounded by a faint halo. Rickettsiae were identified most consistently in small arteries and arterioles of liver, kidney, and leptomeninges, and were localized predominantly to the cytoplasm of endothelial cells and less often within the internal elastic lamella and smooth muscle of the media. This validation of argyrophilic techniques to detect R. rickettsii demonstrates the utility of inexpensive core histochemical methods in the diagnosis of infectious agents in pathology specimens and may have utility in certain resource-limited settings where RMSF is endemic.

Energy-based constitutive modelling of local material properties of canine aortas.

This study aims at determining the in vitro anisotropic mechanical behaviour of canine aortic tissue. We specifically focused on spatial variations of these properties along the axis of the vessel. We performed uniaxial stretch tests on canine aortic samples in both circumferential and longitudinal directions, as well as histological examinations to derive the tissue's fibre orientations. We subsequently characterized a constitutive model that incorporates both phenomenological and structural elements to account for macroscopic and microstructural behaviour of the tissue. We showed the two fibre families were oriented at similar angles with respect to the aorta's axis. We also found significant changes in mechanical behaviour of the tissue as a function of axial position from proximal to distal direction: the fibres become more aligned with the aortic axis from 46° to 30°. Also, the linear shear modulus of media decreased as we moved distally along the aortic axis from 139 to 64 kPa. These changes derived from the parameters in the nonlinear constitutive model agreed well with the changes in tissue structure. In addition, we showed that isotropic contribution, carried by elastic lamellae, to the total stress induced in the tissue decreases at higher stretch ratios, whereas anisotropic stress, carried by collagen fibres, increases. The constitutive models can be readily used to design computational models of tissue deformation during physiological loading cycles. The findings of this study extend the understanding of local mechanical properties that could lead to region-specific diagnostics and treatment of arterial diseases.

Characterization of mechanical properties of lamellar structure of the aortic wall: Effect of aging

Arterial wall tissues are sensitive to their mechanical surroundings and remodel their structure and mechanical properties when subjected to mechanical stimuli such as increased arterial pressure. Such remodeling is evident in hypertension and aging. Aging is characterized by stiffening of the artery wall which is assigned to disturbed elastin function and increased collagen content. To better understand and provide new insight on microstructural changes induced by aging, the lamellar model of the aortic media was utilized to characterize and compare wall structure and mechanical behavior of the young and old human thoracic aortic samples. Such model regards arterial media as two sets of alternating concentric layers, namely sheets of elastin and interlamellar layers. Histological and biaxial tests were performed and microstructural features and stress–strain curves of media were evaluated in young and old age groups. Then using optimization algorithms and hyperelastic constitutive equations the stress–strain curves of layers were evaluated for both age groups. Results indicated slight elevation in the volume fraction of interlamellar layer among old subjects most probably due to age related collagen deposition. Aging indicated substantial stiffening of interlamellar layers accompanied by noticeable softening of elastic lamellae. The general significant stiffening of old samples were attributed to both increase of volume fraction of interlamellar layers and earlier recruitment of collagen fibers during load bearing due to functional loss of elastin within wall lamellae. Mechanical characterization of lamellar structure of wall media is beneficial in study of arterial remodeling in response to alternated mechanical environment in aging and clinical conditions through coupling of wall microstructure and mechanical behavior.

Frequency-modulated atomic force microscopy localises viscoelastic remodelling in the ageing sheep aorta

Age-related aortic stiffening is associated with cardiovascular diseases such as heart failure. The mechanical functions of the main structural components of the aorta, such as collagen and elastin, are determined in part by their organisation at the micrometer length scale. With age and disease both components undergo aberrant remodelling, hence, there is a need for accurate characterisation of the biomechanical properties at this length scale. In this study we used a frequency-modulated atomic force microscopy (FM-AFM) technique on a model of ageing in female sheep aorta (young: ~18 months, old: >8 years) to measure the micromechanical properties of the medial layer of the ascending aorta. The novelty of our FM-AFM method, operated at 30kHz, is that it is non-contact and can be performed on a conventional AFM using the ׳cantilever tune’ mode, with a spatial (areal) resolution of around 1.6μm2. We found significant changes in the elastic and viscoelastic properties within the medial lamellar unit (elastic lamellae and adjacent inter-lamellar space) with age. In particular, there was an increase in elastic modulus (Young; geometric mean (geometric SD)=42.9 (2.26)kPa, Old=113.9 (2.57)kPa, P<0.0001), G′ and G″ (storage and loss modulus respectively) (Young; G′=14.3 (2.26)kPa, Old G′=38.0 (2.57)kPa, P<0.0001; Young; G″=14.5 (2.56)kPa, Old G″=32.8 (2.52)kPa, P<0.0001). The trends observed in the elastic properties with FM-AFM matched those we have previously found using scanning acoustic microscopy (SAM). The utility of the FM-AFM method is that it does not require custom AFM hardware and can be used to simultaneously determine the elastic and viscoelastic behaviour of a biological sample.

Loss of function mutation in LOX causes thoracic aortic aneurysm and dissection in humans

Thoracic aortic aneurysms and dissections (TAAD) represent a substantial cause of morbidity and mortality worldwide. Many individuals presenting with an inherited form of TAAD do not have causal mutations in the set of genes known to underlie disease. Using whole-genome sequencing in two first cousins with TAAD, we identified a missense mutation in the lysyl oxidase (LOX) gene (c.893T > G encoding p.Met298Arg) that cosegregated with disease in the family. Using clustered regularly interspaced short palindromic repeats (CRISPR)/clustered regularly interspaced short palindromic repeats-associated protein-9 nuclease (Cas9) genome engineering tools, we introduced the human mutation into the homologous position in the mouse genome, creating mice that were heterozygous and homozygous for the human allele. Mutant mice that were heterozygous for the human allele displayed disorganized ultrastructural properties of the aortic wall characterized by fragmented elastic lamellae, whereas mice homozygous for the human allele died shortly after parturition from ascending aortic aneurysm and spontaneous hemorrhage. These data suggest that a missense mutation in LOX is associated with aortic disease in humans, likely through insufficient cross-linking of elastin and collagen in the aortic wall. Mutation carriers may be predisposed to vascular diseases because of weakened vessel walls under stress conditions. LOX sequencing for clinical TAAD may identify additional mutation carriers in the future. Additional studies using our mouse model of LOX-associated TAAD have the potential to clarify the mechanism of disease and identify novel therapeutics specific to this genetic cause.

The Ross procedure: biomechanical properties of the pulmonary artery according to aortic valve phenotype.

The aim of this study is to determine whether patients undergoing the Ross procedure with bicuspid aortic valves have pulmonary artery biomechanical properties different from those with tricuspid valves. Thirty-two pulmonary arteries and 20 aortas were obtained from patients undergoing the Ross procedure at the time of surgery, from a cohort of 32 patients. The aortic valve was tricuspid in 5 patients (16%), bicuspid in 18 patients (56%) and unicuspid in 9 patients (28%). Histological analysis and ex vivo equi-biaxial tensile testing completed within 8 hours of surgery were used to evaluate differences in patient groups and between the pulmonary artery and the ascending aorta. There was no difference in thickness among pulmonary arteries when compared according to aortic valve phenotype (P = 0.94). There was no difference in the tensile tissue properties among aortas and pulmonary arteries when compared according to aortic valve phenotype, in either the circumferential or longitudinal axis. When compared according to the main surgical indication, pulmonary artery walls from patients with pure aortic regurgitation were less stiff than their counterparts (aortic regurgitation: 0.055 ± 0.037 MPa, aortic stenosis: 0.103 ± 0.051 MPa, mixed disease: 0.110 ± 0.044 MPa and aortic valve endocarditis: 0.216 ± 0.033 MPa, P = 0.002). There was no difference in the number of elastic lamellae in pulmonary artery specimens from the three different aortic valve phenotypes, as well as in the aortic specimens. No significant differences were observed in the biomechanical properties of pulmonary arteries when compared according to aortic valve phenotype.

The symmetric buckling mode in laminated elastoplastic micro-structures under plane strain

The present work considers lamellar (micro) structures of thin, elastic lamellae embedded in a yielding matrix as a stability problem in the context of the theory of stability and uniqueness of path-dependent systems. The volume ratio of the stiff lamellae to the relatively soft matrix is assumed low enough to initiate a symmetric buckling mode, which is investigated by analytical and numerical means. Using a highly abstracted, incompatible model, a first approach is made, and the principal features of the problem are highlighted. Assuming plane strain deformation, an analytic expression for the bifurcation load of a refined, compatible model is derived for the special case of ideal plasticity and verified by numerical results. The effect of lamella spacing and matrix hardening on the bifurcation load is studied by a finite element unit cell model. Some of the findings for the ideal plastic matrix are shown to also apply for a mildly hardening matrix material. Furthermore, the postbuckling behaviour and the limit load are investigated by simulating a bulk lamella array.

Abstract 300: Pathogenetic Mechanisms of Thoracic Aortic Aneurysm in a Smad4 Mutant Mouse Model: Identification of New Molecular Targets for Pharmacological Therapy

TGFβ signaling have been implicated in the onset of thoracic aortic aneurysms (TAAs). It has been described cases of aortic disease in humans associated to mutations in SMAD4, an intracellular transducer required in the TGFβ canonical pathway. Interestingly, Smad4 homozygotes mice are embryonically lethal and embryonic inactivation of Smad4 in smooth muscle cells leads to early death because of aortic aneurysms. In order to study mechanistic insights and potential translational target of Smad4-dependent aneurysms, we postnatally targeted the gene, using a tamoxifen (TAM) inducible Cre recombinase under the smooth muscle myosin heavy chain promoter. Echocardiography showed a progressive dilatation of aorta and TAA formation. Histological analysis revealed progressive fragmentation of elastic lamellae with increased inflammatory infiltrates at sites of disarrangement. Most of these mice died 4/6 months because of rupture of thoracic aorta. We further characterized the infiltrate of monocytes in the aorta by flow cytometry, finding a significant increase of CD11b + Ly6C hi cells and F4/80 + CD169 + resident macrophages. Thus, we hypothesized that the deletion of Smad4 should activate an immune/inflammatory mechanism in the aorta, which could be in the end responsible for the pathological phenotype. At the molecular level, we actually found a significant increase in one of the noncanonical TGFβ signaling, i.e. the p65 subunit of NFkB, a protein complex involved in regulating responses to different stressors, including cytokines. Among these, we found a selective IL1β upregulation, produced as an inactive precursor and released upon caspase1 cleavage. In turn, caspase1 requires the function of NLRP3 inflammasome, a multiprotein platform that assembles in response to infection and tissue damage and is responsible for activation of inflammatory caspases. Therefore, we also tested NLRP3 mRNA levels and found them to be significantly upregulated. Overall, we demonstrated that selective Smad4 deletion in the SMC of adult mice is sufficient to induce an immune reaction starting from the molecular alterations downstream to TGFβ signaling and culminating in the onset of TAA.