Basement Membrane Alterations Research Articles

Basement Membranes, Brittlestar Tendons, and Their Mechanical Adaptability.

Basement membranes (BMs) are thin layers of extracellular matrix that separate epithelia, endothelia, muscle cells, and nerve cells from adjacent interstitial connective tissue. BMs are ubiquitous in almost all multicellular animals, and their composition is highly conserved across the Metazoa. There is increasing interest in the mechanical functioning of BMs, including the involvement of altered BM stiffness in development and pathology, particularly cancer metastasis, which can be facilitated by BM destabilization. Such BM weakening has been assumed to occur primarily through enzymatic degradation by matrix metalloproteinases. However, emerging evidence indicates that non-enzymatic mechanisms may also contribute. In brittlestars (Echinodermata, Ophiuroidea), the tendons linking the musculature to the endoskeleton consist of extensions of muscle cell BMs. During the process of brittlestar autotomy, in which arms are detached for the purpose of self-defense, muscles break away from the endoskeleton as a consequence of the rapid destabilization and rupture of their BM-derived tendons. This contribution provides a broad overview of current knowledge of the structural organization and biomechanics of non-echinoderm BMs, compares this with the equivalent information on brittlestar tendons, and discusses the possible relationship between the weakening phenomena exhibited by BMs and brittlestar tendons, and the potential translational value of the latter as a model system of BM destabilization.

#2281 SEM for the diagnosis of glomerular diseases (amyloidosis, minimal change, thin membrane, IgA, membranoproliferative and diabetic nephropathy)

Abstract Background and Aims Treating renal diseases often requires ultrastructural details to establish or confirm diagnosis. Ultrastructural information that is not evidenced by optical microscopy includes, e.g., the podocyte foot processes effacement, thinning or alteration of basement membrane, presence of fibrils, Fabri's intracellular inclusions and subepithelial vs. subendothelial of deposits. Unfortunately, transmission electron microscopy (TEM), required to achieve the required resolution, has high costs, and is time-consuming. Here, we report our experience with scanning electron microscopy (SEM) on paraffin sections from standard pathology sections. Method We have retrospectively reanalyzed paraffin sections from renal biopsies of 50 patients with a diagnosis of IgAN, minimal change, diabetes, and amyloidosis. The paraffin sections were available and processed according to standard procedures (fixation, paraffin embedding), with a thickness of 3-4 µm. Sections were deparaffinized, stained with PASM (periodic acid Shiff + methenamine), mounted in aqueous mounting medium and imaged with 40x and 60x objectives. Sections were then unmounted, washed to remove glycerol, dehydrated in absolute alcohol, and air-dried. Finally, they were gold coated (“sputtering”) and analyzed in a scanning electron microscope (Zeiss). Results For the interpretation of SEM images, correlative analysis has been used, imaging precisely the same region of the section using standard optical microscopy and SEM. SEM can quickly identify the typical morphology of podocyte foot processes (Fig. 1A) and the podocyte effacement in minimal change disease (Fig. 1B). SEM can also easily allow the measurement of the thickness of the basement membrane of glomerular capillary walls (Fig. 1C-E) and its thinning in case of thin membrane disease (Fig. 1D) or its increased thickness in the case of diabetes (Fig. 1E). Amyloid fibrils (Fig. 1F) can also be identified in the capillary walls, confirming the diagnosis of amyloidosis Congo-red positive (Fig. 1G). The high resolution of the technique allows us to identify with high reliability the small ruptures of the capillary walls in IgA and membranoproliferative nephritis, with extravasation of erythrocytes and fibrin cloths (Fig. 1H). Mitochondria are well-demarcated, particularly in proximal tubule cells, and often, mitochondrial cristae are visible in samples with adequate fixation (Fig. 1I). Interstitial edema and fibrosis are evident in SEM microscopy (Fig. 1L). At very high magnification, in histological preparations with exceptional fixation, the filtration pores of endothelial cells could be observed (partially visible in Fig. 1E), as well as details such as the brush border of the tubular epithelial cells. The interpretation of the images is not always straightforward as it also requires knowledge of the artifacts induced by the fixation, processing, and inclusion of paraffin, such as, for example, the intracytoplasmic cellular structures containing lipids of the intratubular “foam cells” appear empty. For a correct interpretation of the SEM images, however, the recognition of the nuclei is essential because the chromatin is condensed into clods, especially around the periphery, and inside, some areas appear empty. Conclusion Scanning electron microscopy (SEM) is a powerful tool for the study of nephrological diseases, as it allows you to examine microscopic details at the nanometric scale (not detectable in optical microscopy), providing valuable information for diagnosis, understanding, and management of the disease, the possibility of carrying out Correlative Microscopy. Given the relatively simple and fast preparation of the samples to be examined, the low costs of execution, and the possibility of archiving the preparation in the histotheque for a possible revision of the case, this leads us to think that this method could be considered a valid alternative to transmission electron microscopy (TEM).

ITGAM-mediated macrophages contribute to basement membrane damage in diabetic nephropathy and atherosclerosis

BackgroundDiabetic nephropathy (DN) and atherosclerosis (AS) are prevalent and severe complications associated with diabetes, exhibiting lesions in the basement membrane, an essential component found within the glomerulus, tubules, and arteries. These lesions contribute significantly to the progression of both diseases, however, the precise underlying mechanisms, as well as any potential shared pathogenic processes between them, remain elusive.MethodsOur study analyzed transcriptomic profiles from DN and AS patients, sourced from the Gene Expression Omnibus database. A combination of integrated bioinformatics approaches and machine learning models were deployed to identify crucial genes connected to basement membrane lesions in both conditions. The role of integrin subunit alpha M (ITGAM) was further explored using immune infiltration analysis and genetic correlation studies. Single-cell sequencing analysis was employed to delineate the expression of ITGAM across different cell types within DN and AS tissues.ResultsOur analyses identified ITGAM as a key gene involved in basement membrane alterations and revealed its primary expression within macrophages in both DN and AS. ITGAM was significantly correlated with tissue immune infiltration within these diseases. Furthermore, the expression of genes encoding core components of the basement membrane was influenced by the expression level of ITGAM.ConclusionOur findings suggest that macrophages may contribute to basement membrane lesions in DN and AS through the action of ITGAM. Moreover, therapeutic strategies that target ITGAM may offer potential avenues to mitigate basement membrane lesions in these two diabetes-related complications.

An in vitro model to study immune activation, epithelial disruption and stromal remodelling in inflammatory bowel disease and fistulising Crohn's disease.

At present, preclinical models of inflammatory bowel disease (IBD) are insufficient, limiting translation between research and new therapeutics. This is especially true for fistulising Crohn's disease (CD), as the severe lack of relevant models hinders research progression. To address this, we present in vitro human IBD mucosal models that recapitulate multiple pathological hallmarks of IBD simultaneously in one model system - immune cell infiltration, stromal remodelling and epithelial disruption. Stimulation of models induces epithelial aberrations common in IBD tissue including altered morphology, microvilli abnormalities, claudin gene expression changes and increased permeability. Inflammatory biomarkers are also significantly increased including cytokines and chemokines integral to IBD pathogenesis. Evidence of extracellular matrix remodelling, including upregulated matrix-metalloproteinases and altered basement membrane components, suggests the models simulate pathological stromal remodelling events that closely resemble fistulising CD. Importantly, MMP-9 is the most abundant MMP and mimics the unique localisation observed in IBD tissue. The inflamed models were subsequently used to elucidate the involvement of TNF-α and IFN- γ in intestinal stromal remodelling, in which TNF-α but not IFN- γ induced MMP upregulation, specifically of MMP-3 and MMP-9. Collectively, our results demonstrate the potential of the IBD models for use in preclinical research in IBD, particularly for fistulising CD.

Overexpression of ameloblastin in secretory ameloblasts results in demarcated, hypomineralized opacities in enamel.

Introduction: Developmental defects of the enamel manifest before tooth eruption and include amelogenesis imperfecta, a rare disease of underlying gene mutations, and molar-incisor hypomineralization (MIH), a prevalent disease in children originating from environmental and epigenetic factors. MIH enamel presents as the abnormal enamel marked by loss of translucency, demarcation between the healthy and affected enamel, and reduced mineral content. The pathophysiology of opaque, demarcated enamel lesions is not understood; however, the retention of enamel proteins in the matrix has been suggested. Ameloblastin (Ambn) is an enamel protein of the secreted calcium-binding phosphoproteins (SCPPs) critical for enamel formation. When the Ambn gene is mutated or deleted, teeth are affected by hypoplastic amelogenesis imperfecta. Methods: In this study, enamel formation in mice was analyzed when transgenic Ambn was overexpressed from the amelogenin promoter encoding full-length Ambn. Ambn was under- and overexpressed at six increasing concentrations in separate mouse lines. Results: Mice overexpressing Ambn displayed opaque enamel at low concentrations and demarcated lesions at high concentrations. The severity of enamel lesions increased starting from the inner enamel close to the dentino-enamel junction (DEJ) to span the entire width of the enamel layer in demarcated areas. Associated with the opaque enamel were 17-kDa Ambn cleavage products, a prolonged secretory stage, and a thin basement membrane in the maturation stage. Ambn accumulations found in the innermost enamel close to the DEJ and the mineralization front correlated with reduced mineral content. Demarcated enamel lesions were associated with Ambn species of 17kDa and higher, prolonged secretory and transition stages, a thin basement membrane, and shortened maturation stages. Hypomineralized opacities were delineated against the surrounding mineralized enamel and adjacent to ameloblasts detached from the enamel surface. Inefficient Ambn cleavage, loss of contact between ameloblasts, and the altered basement membrane curtailed the endocytic activity; thus, enamel proteins remained unresorbed in the matrix. Ameloblasts have the ability to distinguish between Ambn concentration and Ambn cleavage products through finely tuned feedback mechanisms. The under- or overexpression of Ambn in murine secretory ameloblasts results in either hypoplastic amelogenesis imperfecta or hypomineralization with opaque or sharply demarcated boundaries of lesions, similar to MIH.

Identification of basement membrane markers in diabetic kidney disease and immune infiltration by using bioinformatics analysis and experimental verification.

Diabetic kidney disease (DKD) is the leading cause of chronic kidney disease worldwide. Basement membranes (BMs) are ubiquitous extracellular matrices which are affected in many diseases including DKD. Here, the authors aimed to identify BM-related markers in DKD and explored the immune cell infiltration in this process. The expression profiles of three datasets were downloaded from the Gene Expression Omnibus database. BM-related differentially expression genes (DEGs) were identified and Kyoto encyclopaedia of genes and genomes pathway enrichment analysis were applied to biological functions. Immune cell infiltration and immune function in the kidneys of patients with DKD and healthy controls were evaluated and compared using the ssGSEA algorithm. The association of hub genes and immune cells and immune function were explored. A total of 30 BM-related DEGs were identified. The functional analysis showed that BM-related DEGs were notably associated with basement membrane alterations. Crucially, BM-related hub genes in DKD were finally identified, which were able to distinguish patients with DKD from controls. Moreover, the authors observed that laminin subunit gamma 1(LAMC1) expression was significantly high in HK2 cells treated with high glucose. Immunohistochemistry results showed that, compared with those in db/m mouse kidneys, the levels of LAMC1 in db/db mouse kidneys were significantly increased. The biomarkers genes may prove crucial for DKD treatment as they could be targeted in future DKD treatment protocols.

The endothelium in lung fibrosis: a core signaling hub in disease pathogenesis?

Pulmonary fibrosis is a progressive chronic lung disease characterized by excessive deposition of extracellular matrix and structural destruction, associated with a severe five year mortality rate. The onset of the disease is thought to be triggered by chronic damage to the alveolar epithelium. Since the pulmonary endothelium is an important component of the alveolar-capillary niche, it is also affected by the initial injury. In addition to ensuring proper gas exchange, the endothelium has critical functional properties, including regulation of vascular tone, inflammatory responses, coagulation and maintenance of vascular homeostasis and integrity. Recent single-cell analyses have shown that shifts in endothelial cell subtypes occur in pulmonary fibrosis. Furthermore, the increased vascular remodeling associated with pulmonary fibrosis leads to deteriorated outcomes for patients, underscoring the importance of the vascular bed in pulmonary fibrosis. To date, the causes and consequences of endothelial and vascular involvement in pulmonary fibrosis are poorly understood. Therefore, it is of great importance to investigate the involvement of endothelial cells and the vascular system in the pathogenesis of the disease. In this review, we will outline the current knowledge on the role of the pulmonary vasculature in pulmonary fibrosis, in terms of abnormal cellular interactions, hyperinflammation, vascular barrier disorders and an altered basement membrane composition. Finally, we will summarize recent advances in extensive therapeutic research and discuss the significant value of novel therapies targeting the endothelium.

Rbpj Deficiency Disrupts Vascular Remodeling via Abnormal Apelin and Cdc42 (Cell Division Cycle 42) Activity in Brain Arteriovenous Malformation.

Brain arteriovenous malformations (bAVM) are characterized by enlarged blood vessels, which direct blood through arteriovenous shunts, bypassing the artery-capillary-vein network and disrupting blood flow. Clinically, bAVM treatments are invasive and not routinely applicable. There is critical need to understand mechanisms of bAVM pathologies and develop pharmacological therapies. We used an in vivo mouse model of Rbpj-mediated bAVM, which develops pathologies in the early postnatal period and an siRNA in vitro system to knockdown RBPJ in human brain microvascular endothelial cells (ECs). To understand molecular events regulated by endothelial Rbpj, we conducted RNA-Seq and chromatin immunoprecipitation-Seq analyses from isolated brain ECs. Rbpj-deficient (mutant) brain ECs acquired abnormally rounded shape (with no change to cell area), altered basement membrane dynamics, and increased endothelial cell density along arteriovenous shunts, compared to controls, suggesting impaired remodeling of neonatal brain vasculature. Consistent with impaired endothelial cell dynamics, we found increased Cdc42 (cell division cycle 42) activity in isolated mutant ECs, suggesting that Rbpj regulates small GTPase (guanosine triphosphate hydrolase)-mediated cellular functions in brain ECs. siRNA-treated, RBPJ-deficient human brain ECs displayed increased Cdc42 activity, disrupted cell polarity and focal adhesion properties, and impaired migration in vitro. RNA-Seq analysis from isolated brain ECs identified differentially expressed genes in mutants, including Apelin, which encodes a ligand for G protein-coupled receptor signaling known to influence small GTPase activity. Chromatin immunoprecipitation-Seq analysis revealed chromatin loci occupied by Rbpj in brain ECs that corresponded to G-protein and Apelin signaling molecules. In vivo administration of a competitive peptide antagonist against the Apelin receptor (Aplnr/Apj) attenuated Cdc42 activity and restored endothelial cell morphology and arteriovenous connection diameter in Rbpj-mutant brain vessels. Our data suggest that endothelial Rbpj promotes rearrangement of brain ECs during cerebrovascular remodeling, through Apelin/Apj-mediated small GTPase activity, and prevents bAVM. By inhibiting Apelin/Apj signaling in vivo, we demonstrated pharmacological prevention of Rbpj-mediated bAVM.

Pathogenesis of melasma

AbstractTo adequately guide future therapies, it is important to elucidate the pathogenesis of melasma. Chronic ultraviolet (UV) radiation exposure, hormonal stimulation, and genetic predisposition play a role in melasma. A widely accepted theory for the pathogenesis of melasma is the increase of melanin through UV, which leads to the upregulation of melanin stimulating receptors (MSR) such as melanocortin‐1 receptors (MC1R), resulting in increased hormone binding and melanin production. Furthermore, there are similarities between the histopathologic presentation of melasma and solar elastosis, a hallmark of photo‐aged skin. These overlaps include altered basement membrane, increased vascularization, and increased mast cell count. There is significant data showing greater degree of solar elastosis in the melasma skin compared to the perilesional skin. Additionally, an etiologic factor in the pathogenesis of melasma is hormonal exposure to estrogen. Melasma has increased prevalence among menstruating women, women on oral contraceptives, and pregnant women. In melasma lesions, studies have shown an increased number of estrogen receptors and progesterone receptors in the dermis and epidermis, respectively. Previously, increased melanogenesis was thought to be the sole contributing factor to the pathogenesis of melasma but more recently, aberrant vasodilation and angiogenesis have been found to play a role. Nitric oxide, a potent vasodilator, was found to be overexpressed at the dermal‐epidermal junction and was hypothesized to contribute to melasma through stimulation of tyrosinase and increasing vascularity. In conclusion, the various mechanisms implicated in melasma include melanocyte dysfunction, solar elastosis, mast cell dysfunction, hormonal influence, vascular transformation, and basement membrane damage.

Sulfur mustard corneal injury is associated with alterations in the epithelial basement membrane and stromal extracellular matrix

Sulfur mustard (SM; bis(2-chloroethyl) sulfide) is a highly reactive bifunctional alkylating agent synthesized for chemical warfare. The eyes are particularly sensitive to SM where it causes irritation, pain, photophobia, and blepharitis, depending on the dose and duration of exposure. In these studies, we examined the effects of SM vapor on the corneas of New Zealand white male rabbits. Edema and hazing of the cornea, signs of acute injury, were observed within one day of exposure to SM, followed by neovascularization, a sign of chronic or late phase pathology, which persisted for at least 28 days. Significant epithelial-stromal separation ranging from ~8–17% of the epithelial surface was observed. In the stroma, there was a marked increase in CD45+ leukocytes and a decrease of keratocytes, along with areas of disorganization of collagen fibers. SM also disrupted the corneal basement membrane and altered the expression of perlecan, a heparan sulfate proteoglycan, and cellular fibronectin, an extracellular matrix glycoprotein. This was associated with an increase in basement membrane matrix metalloproteinases including ADAM17, which is important in remodeling of the basement membrane during wound healing. Tenascin-C, an extracellular matrix glycoprotein, was also upregulated in the stroma 14–28 d post SM, a finding consistent with its role in organizing structural components of the stroma necessary for corneal transparency. These data demonstrate that SM vapor causes persistent alterations in structural components of the cornea. Further characterization of SM-induced injury in rabbit cornea will be useful for the identification of targets for the development of ocular countermeasures.

MO058: Do Renal Biopsies Assist Understanding of Loin Pain Hematuria Syndrome?

Abstract BACKGROUND AND AIMS Loin pain-hematuria syndrome (LPHS) first described in 1967 is a complex and poorly understood rare disease, with a prevalence of 0.012%. It predominantly affects young women in their late 20s and early 30s2. Patients with LPHS experience extreme flank (loin) pain that can be unilateral or bilateral, along with gross or microscopic hematuria. In most cases, the pain is so severe that the patients are forced to quit their jobs, become opiate dependent, need antidepressants, require long term disability and a proportion contemplate committing suicide. Due to inadequate understanding of the pathophysiology of LPHS, the goal of management has been limited to symptomatic relief and pain management. As there are still gaps in our understanding of glomerular hematuria, we opted to assess the histopathological features of kidney biopsies of 14 LPHS patients to evaluate the correlation of glomerular basement membrane (GBM) alterations with hematuria and pain. METHOD In this cross-sectional study, 14 patients with LPHS underwent kidney biopsies. Demographic data (sex, age, ethnicity, weight and height), renal biomarkers (urine blood, urine protein and serum creatinine) and comorbidities were obtained prior to the procedure. The kidney biopsies were investigated under light microscopy, immunofluorescence microscopy and electron microscopy. Descriptive statistics were used to report baseline characteristics and histology findings. RESULTS All 14 patients were female, with median age 37 years. About 78.6% of the patients were Caucasian. None of the 14 patients had an abnormal glomerular filtration rate or an elevated albumin creatinine ratio (>3 mg/mmoL). 11/14 patients had macroscopic hematuria with 7/11 reporting gross hematuria with exacerbations of loin pain. 5/14 patients showed evidence of endothelial injury. Of the 14 patients, 1/14 had an insufficient sample, 1/13 had thick glomerular basement membrane, 1/13 had IgA nephropathy and 11/13 had normal GBM (Table 1). Regardless of GBM thickness, most patients had RBC cells ± casts implying glomerular leak. There was no evidence of podocyte injury. The presence of endothelial cell injury by electron microscopy may suggest a role for vascular damage in the pathogenesis of at least a subgroup of LPHS patients. CONCLUSION The array of histological changes identified in our study offers some understanding of the hematuria component but cannot explain pain as most patients did not have histological evidence of interstitial edema. In conclusion, our findings indicate that there is an urgent need for advanced understanding of molecular mechanisms underlying the heterogeneity of this disease. In our next phase of the study, we intend to compare gene signatures of LPHS patients with patients with isolated hematuria (without pain) to help enhance our understanding of unique genetic predisposing factors to this disease.

Alcohol-Induced Alterations in the Vascular Basement Membrane in the Substantia Nigra of the Adult Human Brain.

The blood–brain barrier (BBB) represents a highly specialized interface that acts as the first line of defense against toxins. Herein, we investigated the structural and ultrastructural changes in the basement membrane (BM), which is responsible for maintaining the integrity of the BBB, in the context of chronic alcoholism. Human post-mortem tissues from the Substantia Nigra (SN) region were obtained from 44 individuals, then grouped into controls, age-matched alcoholics, and non-age-matched alcoholics and assessed using light and electron microscopy. We found significantly less CD31+ vessels in alcoholic groups compared to controls in both gray and white matter samples. Alcoholics showed increased expression levels of collagen-IV, laminin-111, and fibronectin, which were coupled with a loss of BM integrity in comparison with controls. The BM of the gray matter was found to be more disintegrated than the white matter in alcoholics, as demonstrated by the expression of both collagen-IV and laminin-111, thereby indicating a breakdown in the BM’s structural composition. Furthermore, we observed that the expression of fibronectin was upregulated in the BM of the white matter vasculature in both alcoholic groups compared to controls. Taken together, our findings highlight some sort of aggregation or clumping of BM proteins that occurs in response to chronic alcohol consumption.

Effect of Lycopene on Histological Alterations of Basement Membrane & Fibrosis in Renal Interstitial Tissue of Celebrex Induced Albino Rats; An Experimental Study

Objective: To assess the histological alterations in basement membrane and fibrosis in renal interstitium of albino rats due to celebrex with enhancement by lycopene. 
 Study Design: Experimental study.
 Abode of Study: Animal House of Jinnah Postgraduate institute, Karachi,
 Materials and Methods: COX-2 inhibitor and antioxidant medicines were used in this research work. These medications were orally administered in 40 male albino rats weighing 200-220gm for experimentation. Rats were housed in separate pens at 23ºC. Rats were arranged into 4 groups including control horde and three experimental hordes. The medications were dose up orally by gastric tube daily for one month. At completion of experiment, animals were dissected and tissues were well-preserved for staining.
 Results: In second horde PAS stained kidney segments showed disrupted basement membrane of distended proximal convoluted tubules & ill-defined brush border and fibrosis in renal interstitium, but 3rd horde had intact basement membrane & well-define brush border at the luminal surface of proximal tubular epithelium and there was mild fibrosis in renal interstitium.
 Conclusion: This study divulges that lycopene convalesce the disrupted basement membrane and fibrosis in second horde.

Evaluation of ultrastructural alterations of glomerular basement membrane and podocytes in glomeruli by low-vacuum scanning electron microscopy

BackgroundLow-vacuum scanning electron microscopy (LV-SEM) is applied to diagnostic renal pathology.MethodsTo demonstrate the usefulness of LV-SEM and to clarify the optimal conditions of pathology samples, we investigated the alterations of glomerular basement membrane (GBM) and podocytes in control and experimental active Heymann nephritis (AHN) rats by LV-SEM.ResultsOn week 15 following induction of AHN, spike formation on GBM with diffuse deposition of IgG and C3 developed. Using LV-SEM, diffuse crater-like protrusions were clearly noted three-dimensionally (3D) on surface of GBM in the same specimens of light microscopy (LM) and immunofluorescence (IF) studies only after removal coverslips or further adding periodic acid-silver methenamine (PAM) staining. These 3D ultrastructural findings of GBM surface could be detected in PAM-stained specimens by LV-SEM, although true GBM surface findings could not be obtained in acellular glomeruli, because some subepithelial deposits remained on surface of GBM. Adequate thickness was 1.5–5 μm for 10% formalin-fixed paraffin-embedded (FFPE) and 5–10 μm for the unfixed frozen sections. The foot processes and their effacement of podocytes could be observed by LV-SEM using 10%FFPE specimens with platinum blue (Pt-blue) staining or double staining of PAM and Pt-blue. These findings were obtained more large areas in 2.5% glutaraldehyde-fixed paraffin-embedded (2.5%GFPE) specimens.ConclusionOur findings suggest that LV-SEM is a useful assessment tool for evaluating the alterations of GBM and podocytes in renal pathology using routine LM and IF specimens, as well as 2.5%GFPE specimens.

Hemicentin-1 is an essential extracellular matrix component of the dermal\u2013epidermal and myotendinous junctions

The extracellular matrix architecture is composed of supramolecular fibrillar networks that define tissue specific cellular microenvironments. Hemicentins (Hmcn1 and Hmcn2) are ancient and very large members (> 600 kDa) of the fibulin family, whose short members are known to guide proper morphology and functional behavior of specialized cell types predominantly in elastic tissues. However, the tissue distribution and function of Hemicentins within the cellular microenvironment of connective tissues has remained largely unknown. Performing in situ hybridization and immunofluorescence analyses, we found that mouse Hmcn1 and Hmcn2 show a complementary distribution throughout different tissues and developmental stages. In postnatal dermal–epidermal junctions (DEJ) and myotendinous junctions (MTJ), Hmcn1 is primarily produced by mesenchymal cells (fibroblasts, tenocytes), Hmcn2 by cells of epithelial origin (keratinocytes, myocytes). Hmcn1−/− mice are viable and show no overt phenotypes in tissue tensile strength and locomotion tests. However, transmission electron microscopy revealed ultrastructural basement membrane (BM) alterations at the DEJ and MTJ of Hmcn1−/− mice, pointing to a thus far unknown role of Hmcn1 for BM and connective tissue boundary integrity.

The pathogenesis of melasma and implications for treatment.

Melasma is a complex and poorly understood disorder, with high rates of treatment failure and recurrences. We aimed to review the current knowledge of the pathogenesis of melasma and apply this knowledge to clinical implications on relevant therapeutic interventions. A systematic PubMed search was performed using the search term "((melasma[Text Word]) OR facial melanosis[Text Word]) AND (pathogenesis OR causality[MeSH Terms])" for articles published between 1990 and 2020. Included articles were then evaluated by two authors and assessed for relevant pathomechanistic pathways, after which they were divided into groups with minimal overlap. We then reviewed current treatment modalities for melasma and divided them according to the involved pathomechanistic pathway. A total of 309search results were retrieved among which 76 relevant articles were identified and reviewed. Five main pathomechanisms observed in melasma were identified: (1) melanocyte inappropriate activation; (2) aggregation of melanin and melanosomes in dermis and epidermis; (3a) increased mast cell count and (3b) solar elastosis; (4) altered basement membrane; and (5) increased vascularization. Treatment modalities were then divided based on these five pathways and detailed in 6 relevant tables. The pathophysiology of melasma is multifactorial, resulting in treatment resistance and high recurrence rates. This wide variety of pathomechanisms should ideally be addressed separately in the treatment regimen in order to maximize results.

Mild traumatic brain injury induces microvascular injury and accelerates Alzheimer-like pathogenesis in mice

IntroductionTraumatic brain injury (TBI) is considered as the most robust environmental risk factor for Alzheimer’s disease (AD). Besides direct neuronal injury and neuroinflammation, vascular impairment is also a hallmark event of the pathological cascade after TBI. However, the vascular connection between TBI and subsequent AD pathogenesis remains underexplored.MethodsIn a closed-head mild TBI (mTBI) model in mice with controlled cortical impact, we examined the time courses of microvascular injury, blood–brain barrier (BBB) dysfunction, gliosis and motor function impairment in wild type C57BL/6 mice. We also evaluated the BBB integrity, amyloid pathology as well as cognitive functions after mTBI in the 5xFAD mouse model of AD.ResultsmTBI induced microvascular injury with BBB breakdown, pericyte loss, basement membrane alteration and cerebral blood flow reduction in mice, in which BBB breakdown preceded gliosis. More importantly, mTBI accelerated BBB leakage, amyloid pathology and cognitive impairment in the 5xFAD mice.DiscussionOur data demonstrated that microvascular injury plays a key role in the pathogenesis of AD after mTBI. Therefore, restoring vascular functions might be beneficial for patients with mTBI, and potentially reduce the risk of developing AD.

Could basement membrane alterations, resembling micro-wounds at the dermo-epidermal junction in psoriatic non-lesional skin, make the skin susceptible to lesion formation?

Current data suggest that tissue microenvironment control immune functions. Therefore, understanding the tissue environment in which immune activation occurs will enhance our capability to interfere with abnormal immune pathology. Here, we argue that studying the constitutively abnormal functions of clinically uninvolved psoriatic skin in patients with plaque type psoriasis is very important to better understand psoriasis pathobiology, because non-lesional skin provides the tissue environment in which the psoriatic lesion develops. A key question in psoriasis is what initiates the abnormal, uncontrolled immune activation in the first place and the answer may lie in the skin. In light of this concept, we summarize abnormalities at the dermal-epidermal junction region which shows a special "non-healing-like" micro-wound phenotype in the psoriatic non-lesional skin that may act as a crucial susceptibility factor in the development of the disease.

Clinicopathological Analysis of Acute/Active Antibody-Mediated Rejection in Renal Allografts According to the Banff 2013 Classification

Aim: This study evaluated the clinicopathological findings of acute/active antibody-mediated rejection (AABMR) according to the Banff 2013 classification. Methods: We analyzed 345 biopsies of 269 kidney transplant recipients. Pathological AABMR (PAABMR) was defined as histological evidence of acute tissue injury and endothelial injury by light microscopy regardless of donor-specific antibodies (DSAs). Results: Among the 345 biopsies, 29 (8.4%) were diagnosed as PAABMR. The mean g score was 1.17 ± 0.60, the mean ptc score was 1.97 ± 1.32, and DSA positivity was found in 69% of PAABMR. The mean duration after transplantation was 22.9 ± 26.7 months. Among 3 groups (DSA-high, mean fluorescence intensity (MFI) ≥ 5,000; DSA-low, MFI < 5,000 to ≥1,000; below cutoff), ABO incompatibility in DSA-high was significantly lower and second transplantation in DSA-high was significantly higher. We found 83% of PAABMR by the protocol biopsy (subclinical AABMR [SAABMR]). The short-term clinical and light microscopical changes in 8 cases of SAABMR did not show worsening during follow-up period (9–24 months). However, ultrastructural finding, including glomerular endothelial swelling, subendothelial electron-lucent widening, and early glomerular basement duplication, were found by electron microscopy (EM) in the first biopsies, and half of the SAABMR cases developed de novo circular peritubular capillary multilayering in the follow-up biopsies. Conclusion: PAABMR was mainly found by the protocol biopsy. The short-term follow-up of SAABMR patients did not show worsening clinically and light microscopically, but ultrastructural examination by EM was useful to detect early lesions of endothelial injury and progression of glomerular and peritubular capillary basement membrane alterations.

Setting of reliable immunohistochemical criteria for the recurrence of nodular basal cell carcinoma

Introduction. Basal cell carcinoma (BCC) is one of the most prevalent skin neoplasms with increasing incidence. The grade of BCC malignancy is highly variable and depends on the invasiveness and recur-rence potential. The study was aimed at identification of immunohistochemical (IHC) determinants of BCC recurrence. Materials and methods. The comparative study encompassed 10 cases of primary BCC and 10 cases of recurrent BCC. The panel of immunohistochemical targets included p53, CK8/18, Bcl-2, CK19, Collagen type IV, Desmin, CD8, Ki-67, Vimentin, VEGFR, EGFR and AR. Results. Diffuse expression of vimentin (characteristic of both primary and recurrent BCCs and clearly indicating the border between the tumor stroma and the surrounding dermis) in the recurrent tumors was twice as strong as in the primary tumors. Immunohistochemistry for collagen type IV revealed different nature of the basement membrane alterations in the primary and recurrent tumors. A two-fold increase in the intensity of angiogenesis observed in the recurrent tumors was accompanied by a more than two-fold significant increase in the androgen receptor protein expression. Conclusion.Increasing grade of BCC malignancy is associated with the immunohistochemically revealed reinforcement of the stromal and vascular components of the tumor, as well as progressive destruction of the basement membrane along with the increased expression of androgen receptor protein by tumor cells. Keywords: basal cell carcinoma, recurrence, immunohistochemistry