Ciprofloxacin accelerates aortic enlargement and promotes dissection and rupture in Marfan mice.

OBJECTIVE: Aortic aneurysm and dissection are major life-threatening complications of Marfan syndrome. Avoiding factors that promote aortic damage is critical in managing the care of these patients. Findings from clinical and animal studies raise concerns regarding fluoroquinolone use in patients at risk for aortic aneurysm and dissection. Therefore, we examined the effects of ciprofloxacin on aortic aneurysm and dissection development in Marfan mice. METHODS: Eight-week-old Marfan mice (Fbn1C1041G/+) were given ciprofloxacin (100 mg/kg/d; n = 51) or vehicle (n = 59) for 4 weeks. Mice were monitored for 16 weeks. Aortic diameters were measured by using ultrasonography, and aortic structure was examined by using histopathologic and immunostaining analyses. RESULTS: Vehicle-treated Fbn1C1041G/+ mice showed progressive aortic enlargement, with aortic rupture occurring in 5% of these mice. Compared with vehicle-treated Fbn1C1041G/+ mice, ciprofloxacin-treated Fbn1C1041G/+ mice showed accelerated aortic enlargement (P = .01) and increased incidences of aortic dissection (25% vs 47%, P = .03) and rupture (5% vs 25%, P = .005). Furthermore, ciprofloxacin-treated Fbn1C1041G/+ mice had higher levels of elastic fiber fragmentation, matrix metalloproteinase expression, and apoptosis than did vehicle-treated Fbn1C1041G/+ mice. CONCLUSIONS: Ciprofloxacin accelerates aortic root enlargement and increases the incidence of aortic dissection and rupture in Marfan mice, partially by suppressing lysyl oxidase expression and further compromising the inherited defect in aortic elastic fibers. Our findings substantiate that ciprofloxacin should be avoided in patients with Marfan syndrome.

Rabbit Elastase Aneurysm: Imaging and Histology Correlates for Inflammation and Healing.

OBJECTIVE: Aneurysmal subarachnoid hemorrhage remains a devastating event with poorly understood pathophysiology. Previous studies have suggested that aneurysm wall inflammation may play a part in the development and potential rupture of aneurysms. The rabbit elastase aneurysm model is a well-established model, which produces aneurysms closely mimicking human cerebral aneurysms in flow dynamics and histopathology. The primary aim of this study was to correlate inflammatory changes after aneurysm formation using sequential vessel wall imaging with histopathologic analysis. A secondary aim was to evaluate the potential effect of gender and anti-inflammatory treatment with aspirin on this inflammatory response. METHODS: Twenty-seven New Zealand rabbits underwent surgery to create an aneurysm using elastase infusion at the right common carotid artery origin. Vessel wall imaging and histopathologic analysis was obtained at different time points after aneurysm creation. The rabbits were also randomized by gender and to treatment groups with or without aspirin. RESULTS: Histopathologic analysis revealed 3 distinct phases after aneurysm formation. These phases were an initial inflammatory phase, followed by a regeneration phase, and finally a connective tissue deposition phase. Vessel wall imaging demonstrated 2 distinct imaging patterns. No appreciable differences were seen in histology or imaging when comparing gender or treatment with aspirin. CONCLUSIONS: Inflammatory changes induced by the rabbit elastase aneurysm model can be correlated with histopathologic findings and observed on noninvasive vessel wall imaging. This may provide a method to study the inflammatory pathway as it pertains to aneurysmal development and subsequent rupture.

The fibrillin microfibril/elastic fibre network: A critical extracellular supramolecular scaffold to balance skin homoeostasis.

Supramolecular networks composed of fibrillins (fibrillin-1 and fibrillin-2) and associated ligands form intricate cellular microenvironments which balance skin homoeostasis and direct remodelling. Fibrillins assemble into microfibrils which are not only indispensable for conferring elasticity to the skin, but also control the bioavailability of growth factors targeted to the extracellular matrix architecture. Fibrillin microfibrils (FMF) represent the core scaffolds for elastic fibre formation, and they also decorate the surface of elastic fibres and form independent networks. In normal dermis, elastic fibres are suspended in a three-dimensional basket-like lattice of FMF intersecting basement membranes at the dermal-epidermal junction and thus conferring pliability to the skin. The importance of FMF for skin homoeostasis is illustrated by the clinical features caused by mutations in the human fibrillin genes (FBN1, FBN2), summarized as "fibrillinopathies." In skin, fibrillin mutations result in phenotypes ranging from thick, stiff and fibrotic skin to thin, lax and hyperextensible skin. The most plausible explanation for this spectrum of phenotypic outcomes is that FMF regulate growth factor signalling essential for proper growth and homoeostasis of the skin. Here, we will give an overview about the current understanding of the underlying pathomechanisms leading to fibrillin-dependent fibrosis as well as forms of cutis laxa caused by mutational inactivation of FMF-associated ligands.

Aplasia cutis congenita of the scalp: Histopathologic features and clinicopathologic correlation in a case series.

BACKGROUND: Aplasia cutis congenita (ACC) is a rare and heterogeneous disorder characterized by congenital absence of skin. The scalp is the most commonly affected site and lesions may overlie deeper ectodermal abnormalities. The exact etiology is still unknown, and histopathologic features are poorly defined. METHODS: A series of 10 cases from nine patients was analyzed to characterize the clinicopathologic spectrum and age-related changes of ACC of the scalp. Hematoxylin and eosin, S100, Elastica van Gieson, and Weigert elastic stains were performed, and clinical information was retrieved from archived medical files. RESULTS: Patient ages ranged from 1 day to 39 years (median 57 months). All cases resembled deep-reaching scars with almost complete loss of all adnexal structures. Isolated residual hair follicles were present in 8/10 and sweat glands and ducts in 2/10 cases. The subcutis was thinned or absent. Elastic fibers were always more fragmented than in normal tissue, and the thickness and density increased over time. There was no gain of adnexal structures with increasing age. CONCLUSIONS: ACC represents a congenital scarring alopecia with permanent loss of skin appendages. Histopathologic changes resemble a deep-reaching scar with fragmented elastic fibers and differentiate ACC from all other forms of non-traumatic congenital alopecias.

A comprehensive map of human elastin cross-linking during elastogenesis.

Elastin is an essential structural protein in the extracellular matrix of vertebrates. It is the core component of elastic fibers, which enable connective tissues such as those of the skin, lungs or blood vessels to stretch and recoil. This function is provided by elastin's exceptional properties, which mainly derive from a unique covalent cross-linking between hydrophilic lysine-rich motifs of units of the monomeric precursor tropoelastin. To date, elastin's cross-linking is poorly investigated. Here, we purified elastin from human tissue and cleaved it into soluble peptides using proteases with different specificities. We then analyzed elastin's molecular structure by identifying unmodified residues, post-translational modifications and cross-linked peptides by high-resolution mass spectrometry and amino acid analysis. The data revealed the presence of multiple isoforms in parallel and a complex and heterogeneous molecular interconnection. We discovered that the same lysine residues in different monomers were simultaneously involved in various cross-link types or remained unmodified. Furthermore, both types of cross-linking domains, Lys-Pro and Lys-Ala domains, participate not only in bifunctional inter- but also in intra-domain cross-links. We elucidated the sequences of several desmosine-containing peptides and the contribution of distinct domains such as 6, 14 and 25. In contrast to earlier assumptions proposing that desmosine cross-links are formed solely between two domains, we elucidated the structure of a peptide that proves a desmosine formation with participation of three Lys-Ala domains. In summary, these results provide new and detailed insights into the cross-linking process, which takes place within and between human tropoelastin units in a stochastic manner.

SOPH syndrome in three affected individuals showing similarities with progeroid cutis laxa conditions in early infancy.

Individuals affected with autosomal recessive cutis laxa type 2B and 3 usually show translucent skin with visible veins and abnormal elastic fibers, intrauterine and/or postnatal growth restriction and a typical triangular facial gestalt. Here we describe three unrelated individuals in whom such a cutis laxa syndrome was suspected, especially after electron microscopy revealed immature and less dense dermal elastic fibers in one of them. However, one of these children also displayed optic atrophy and two hypogammaglobulinemia. All had elevated liver enzymes and acute liver failure during febrile episodes leading to early demise in two of them. The only surviving patient had been treated with immunoglobulins. Through exome sequencing we identified mutations in NBAS, coding for a protein involved in Golgi-to-ER transport. NBAS deficiency causes several rare conditions ranging from isolated recurrent acute liver failure to a multisystem disorder mainly characterized by short stature, optic nerve atrophy and Pelger-Huët anomaly (SOPH). Since we subsequently verified Pelger-Huët anomaly in two of the patients the diagnosis SOPH syndrome was unequivocally proven. Our data show that SOPH syndrome can be regarded as a differential diagnosis for the progeroid forms of cutis laxa in early infancy and that possibly treatment of the hypogammaglobulinemia can be of high relevance for the prognosis.

Stereological analysis of elastic fibers of the corpus cavernosum of rats during the aging process

Abstract Purpose To evaluate changes in the quantity of elastic fibers in the corpora cavernosa of rats during the natural aging process, and to assess the degree of this change by determining volumetric density (Vv) at different ages via stereological analysis. Methods Forty-eight rats, raised under similar conditions, were subjected to the natural aging process and divided into four groups (G1 to G4), according to age at the time of penectomy (6, 9, 12, and 24 months, respectively). Histological sections of the middle segment of the penis were stained with Weigert's resorcin-fuchsin, and the volumetric density (Vv) of elastic fibers of the corpora cavernosa were determined via stereological analysis. Results There were no statistically significant differences in Vv among groups G1, G2, and G3. These three groups were therefore considered as a single group. The mean Vv of this group showed a statistically significant reduction compared to that of G4 (0.16 vs. 0.11, p<0.05). Conclusion Natural aging in rats was responsible for a reduction in volumetric density of elastic fibers of the corpora cavernosa (approximately 30% decrease in Vv) during senescence.

[Venous invasions in colonic adenocarcinoma: Value of elastic stain]. / Invasions veineuses dans l'adénocarcinome colique : intérêt d'une coloration des fibres élastiques.

The aim of our study was to assess the value of Elastic stain in the diagnosis of venous invasion (VI) in colonic adenocarcinoma. MATERIAL AND METHODS: All patients who undergone surgery for colonic adenocarcinoma at the University Hospital of Amiens, between 2004 and 2007, were included. Hematein-phloxin-saffron (HPS) stained slides of colectomy specimens were reviewed by two pathologists. Tumor blocks were stained with Elastic Stain (Roche - Ventana®). The presence or absence of VI, their number and localization were correlated with overall survival. RESULTS: Two hundred and thirty-one cases were investigated and 3274 slides were examined. VI were more often diagnosed by Elastic Stain than HPS stain (66% vs. 40%). Ninety percent of VI were revealed within the first 6 HPS slides, and from the first 5 in Elastic Stain. The presence of VI revealed by Elastic Stain and/or HPS was significantly associated with decreased overall survival in multivariate analysis (P=0.029), especially for stage IIA tumors (P=0.016). Tumor differentiation (P=0.006) and pTNM stage (P=0.001) were also independent prognostic factors. The localization and the number of VI were not prognostic factors. CONCLUSION: Our study confirms the prognostic value of VI, revealed by an elastic stain, in colonic adenocarcinoma. A systematic elastic stain of all tumor blocks (number at least 5) could be considered in the future, during pathological examination of colectomy for adenocarcinoma.

Ultrastructural aspects of pseudoxanthoma elasticum

Abstract: We report the ultrastructural findings in a case of a 72-year-old black woman with confluent yellowish papules in the cervical region. She had no comorbidities. Ophthalmological examination, electrocardiogram, and echocardiogram were normal. Hematoxylin-eosin staining of the affected skin showed strong alterations in the mid-dermis with irregular clumps of eosinophilic material and loss of the normal parallel arrangement of collagen bundles. Orcein staining revealed that the elastic fibers lost their normal linear configuration, showing clump fragmentation, sometimes forming square structures. Transmission electron microscopy showed aberrant elastic fibers with an irregular outline and heterogenic inner structures. We also observed small elastic fibers. Collagen fibers showed a normal structure with irregular distribution. Scanning electron microscopy revealed important disorganization of collagen fibers and small stone-like deposits measuring around 5 µm associated with bigger structures ranging from 10-16 µm. Higher magnification revealed that these small stone-like structures were sometimes polyhedral-shaped or squared.

Idiopathic atrophoderma of Pasini and Pierini: A case study of collagen and elastin texture by multiphoton microscopy.

BACKGROUND: The diagnosis of idiopathic atrophoderma of Pasini and Pierini (IAPP) relies on typical clinical features, particularly distinctive pigmented ovular/round depressed plaques. Histologic examination often reveals no obvious changes, but patterns of collagen distribution, using multiphoton imaging and second harmonic generation can help track hidden details of tissue organization contributing to atrophy. OBJECTIVE: To identify histologic features that distinguish IAPP from unaffected skin. METHODS: Eleven patients were included for conventional analyses. Masson trichrome- and Unna-Tanzer orcein-stained sections were evaluated using automated morphometry. Hematoxylin-eosin-stained sections were analyzed by multiphoton imaging using 2-photon excited fluorescence and second harmonic generation. RESULTS: No abnormalities were found under light microscopy or by automated quantification. Multiphoton imaging revealed no difference in optical density of either collagen or elastic fibers in lesioned and unaffected skin; however, horizontal collagen fiber organization in lesion specimens increased toward the lower dermis, whereas elastic fibers featured greater disorganization within the upper dermis. LIMITATIONS: The low number of patients evaluated. CONCLUSION: The atrophic appearance of IAPP lesions reflects changes in organization, but not in collagen and elastic tissue content. Minute organizational differences that are imperceptible to the experienced pathologist and undetectable by automated analyses were revealed by multiphoton analyses, particularly second harmonic generation, in association with texture analyses.

Automated quantification of three-dimensional organization of fiber-like structures in biological tissues.

Fiber-like structures are prevalent in biological tissues, yet quantitative approaches to assess their three-dimensional (3D) organization are lacking. We develop 3D directional variance, as a quantitative biomarker of truly 3D fibrillar organization by extending the directional statistics formalism developed for describing circular data distributions (i.e. when 0° and 360° are equivalent) to axial ones (i.e. when 0° and 180° are equivalent). Significant advantages of this analysis include its time efficiency, sensitivity and ability to provide quantitative readouts of organization over different size scales of a given data set. We establish a broad range of applications for this method by characterizing collagen fibers, neuronal axons and fibroblasts in the context of cancer diagnostics, traumatic brain injury and cell-matrix interactions in developing engineered tissues. This method opens possibilities for unraveling in a sensitive, and quantitative manner the organization of essential fiber-like structures in tissues and ultimately its impact on tissue function.

Epithelial-mesenchymal interaction mechanisms leading to the over-expression of neprilysin are involved in the UVB-induced formation of wrinkles in the skin.

In clinical studies, the formation of facial wrinkles has been closely linked to the loss of elastic properties of the skin. Repetitive UVB irradiation of animal skin at suberythemal doses significantly reduces its elastic properties, resulting in the formation of wrinkles. That also elicits a marked alteration in the three-dimensional structure of elastic fibres, which is closely associated with a subsequent reduction in the elastic properties of the skin. While UVB irradiation stimulates the activity of skin fibroblast-derived elastase in the dermis, a synthetic inhibitor specific for skin fibroblast-derived elastase as well as an extract of Zingiber officinale (L.) Rose capable of inhibiting skin fibroblast-derived elastase, but not neutrophil elastase, prevented wrinkle formation in our studies of animal and human facial skin, respectively. The close interrelationship among wrinkle formation, elastic properties and elastic fibre linearity is revealed by the effects of different concentrations of the elastase inhibitor, which indicates that enhanced elastase activity by dermal fibroblasts plays a pivotal role in the UVB wrinkling mechanism. Fortunately, we were able to identify human skin fibroblast-derived elastase as the previously known enzyme neprilysin/neutral endopeptidase. Using both a UVB-conditioned medium assay and a co-culture system, we characterized the epithelial-mesenchymal interaction between keratinocytes and fibroblasts which leads to increased expression of neprilysin at the transcriptional, translational and enzymatic levels. Our results demonstrate that interleukin-1α and granulocyte-macrophage colony-stimulating factor are intrinsic cytokines secreted by UVB-exposed keratinocytes that stimulate the expression of neprilysin by skin fibroblasts.

Ultrastructural evaluation of gingival connective tissue in hereditary gingival fibromatosis.

OBJECTIVE: To describe the ultrastructural features of hereditary gingival fibromatosis (HGF) in affected family members and compare microscopic findings with normal gingival (NG) tissue. STUDY DESIGN: Gingival tissue samples from nine patients with HGF from five unrelated families were evaluated by transmission electron microscopy. Nine NG tissue samples were used for comparison. RESULTS: Areas containing collagen fibrils forming loops and folds were observed in both groups, whereas oxytalan fibers were frequently identified in the HGF group. The diameter of collagen fibrils and the interfibrillar space among them were more uniform in the NG group than in the HGF group. Fibroblasts were the most common cells found in both the HGF and NG groups and exhibited enlarged, rough endoplasmic reticulum, mitochondria with well-preserved crests, conspicuous nucleoli, and euchromatic chromatin. Other cells, such as mast cells, plasma cells, and macrophages, were also observed. CONCLUSIONS: HGF tissues had ultrastructural characteristics that were very similar to those of NG tissues. Oxytalan fibers were observed more frequently in the HGF samples than in the NG samples. Other studies of HGF in patients from different families should be performed to better understand the pathogenesis of this hereditary condition.

Osteogenesis imperfecta: Ultrastructural and histological findings on examination of skin revealing novel insights into genotype-phenotype correlation.

Osteogenesis imperfecta (OI) is a heterogeneous group of inherited disorders of bone formation, resulting in low bone mass and an increased propensity to fracture. Over 90% of patients with OI have a mutation in COL1A1/COL1A2, which shows an autosomal dominant pattern of inheritance. In-depth phenotyping and in particular, studies involving manifestations in the skin connective tissue have not previously been undertaken in OI. The aims of the study were to perform histological and ultrastructural examination of skin biopsies in a cohort of patients with OI; to identify common and distinguishing features in order to inform genotype-phenotype correlation; and to identify common and distinguishing features between the different subtypes of OI. As part of the RUDY (Rare Diseases in Bone, Joints and/or Blood Vessels) study, in collaboration with the NIHR Rare Diseases Translational Research Collaboration, we undertook a national study of skin biopsies in patients with OI. We studied the manifestations in the skin connective tissue and undertook in-depth clinical and molecular phenotyping of 16 patients with OI. We recruited 16 patients: analyses have shown that in type 1 collagen mutation positive patients (COL1A1/ COL1A2) (n-4/16) consistent findings included: variable collagen fibril diameter (CFD) and presence of collagen flowers. Histological examination in these patients showed an increase in elastic fibers that are frequently fragmented and clumped. These observations provide evidence that collagen flowers and CFD variability are consistent features in OI due to type 1 collagen defects and reinforce the need for accurate phenotyping in conjunction with genomic analyses.

Histologic comparison between the internal mammary artery and the deep inferior epigastric artery and clinical implications for microsurgical breast reconstruction.

BACKGROUND: The internal mammary artery (IMA) is one of the most popular recipients for microsurgical breast reconstruction. However, it is often separated into sleeve-like layers when it is handled. This study tried to explain this unique behaviour of the IMA through histologic observation. METHODS: Nine pairs of IMAs and DIEAs were harvested and subject for haematoxylin-eosin and Verhoeff's elastic staining. Thickness of the tunica media and the number of elastic lamellae were compared. Samples of the IMA, the DIEA, and the thoracodorsal artery from another patient were observed through the transmission electron microscope to further show the structural differences. RESULTS: The most notable difference was presence of multiple elastic lamellae in tunica media in the IMAs, which was barely present in the DIEAs. The mean number of elastic lamellae was 9.2 in the IMA group and 1.0 in the DIEA group (p < 10(-9)). A transmission electron microscope showed that the tunica media of the DIEA and the TDA was densely packed with smooth muscle cells, while the muscle cells distributed sparsely in the IMA. CONCLUSIONS: The IMA is an elastic artery which is characterised by multiple layers of elastic lamellae while relatively lacking in smooth muscle cells. The wall of the IMA is easily dissected between the tunica media and the adventitia, or at the outer 1/3 of the tunica media. The inner structure is easily torn if microsutures do not engage the tunica adventitia.

[The structure of the internal carotid artery wall in pathological tortuosity].

OBJECTIVE: to study a change in the content of main components of the internal carotid artery (ICA) wall in pathological tortuosity (PT) resulting from fibromuscular dysplasia, using immunohistochemistry and confocal laser scanning microscopy. MATERIAL AND METHODS: Immunohistochemical (IHC) analysis using antibodies to elastin, collagen types I and III, and smooth muscle actin was made. The levels of elastin and matrix metalloproteinase 9 (MMP-9) were determined by confocal laser scanning microscopy. The relative area of expression and the area of co-location of these markers were measured. RESULTS: IHC examination of the expression of elastin revealed that the patients with PT of ICA had its higher content than the controls, but they were observed to have fiber fragmentation. Comparison of collagen types I and III expressions showed no significant differences between the groups. The found significantly lower smooth muscle actin expression in the patients with PT of ICA than in the controls was suggestive of the decreased levels of smooth muscle cells. Confocal microscopy analysis showed high elastin and low MMP-9 expressions in the control group and, on the contrary, low elastin and high proteinase levels in the PT group (р<0.05). CONCLUSION: One of the causes of PT is impairment in vascular elastic properties due to the destruction of elastic fibers and to their fragmentation, as well as to the decreased count of smooth muscle cells, which in turn causes enhanced MMP-9 activity and tissue matrix degradation.

Structural changes of dilated pelvic ureters in adults.

PURPOSE: To perform a quantitative assessment of different types of pelvic ureter tissues with chronic dilation in adults, using stereological methods. METHODS: We analyzed fragments of dilated pelvic ureters obtained from 6 patients aged between 35 and 67 years (mean, 46 years) who underwent ureteral reimplantation surgery for different reasons. The obstruction duration ranged from 27 to 180 days (mean, 93 days). The control group included fragments of normal pelvic ureters obtained during nephrectomy in 7 kidney transplant donors (age: range, 25-51 years; mean, 36 years). The volumetric density of collagen in the ureter, elastic fibers, and smooth muscle fibers was assessed. RESULTS: The volumetric density (Vv) of collagen showed no significant difference between the two groups (control: 45.3 ± 6.1; dilated: 40.8 ± 6.9; P = 0.23). A statistically significant increase in Vv of elastic fibers was observed in the dilated ureters (control: 18.4 ± 1.2; dilated: 24.6 ± 5.4; P = 0.03). A statistically significant increase in the Vv of smooth muscle fibers was observed in the dilated ureter (control: 42.0 ± 6.0; dilated: 56.2 ± 6.1; P = 0.001). CONCLUSION: The chronically dilated pelvic ureters had significantly more elastin and smooth muscle contents than the controls.

Hajek revisited: a histological examination of the quadrangular membrane.

OBJECTIVE: The predictability of laryngeal cancer spread is due in part to connective tissue membranes. These membranes function as barriers to cancer and divide the larynx into subunits. The field of laryngeal conservation surgery is based on these concepts. The quadrangular membrane plays an important role, hindering the lateral spread of cancer in the larynx. The composition of this membrane has not been well described in the literature. In this study, we examine basic characteristics of the quadrangular membrane using histological techniques. METHODS: Whole organ sections of the larynx were used. These sections were examined under a microscope with stains specific for collagen and elastin. RESULTS: Examination of the sections revealed that the quadrangular membrane is made up of closely woven undulating collagen and elastic fibers. CONCLUSION: The quadrangular membrane is a fibroelastic structure providing a barrier to cancer spread.