Fibrosis Is a Basement Membrane-Related Disease in the Cornea: Injury and Defective Regeneration of Basement Membranes May Underlie Fibrosis in Other Organs.

Every organ develops fibrosis that compromises functions in response to infections, injuries, or diseases. The cornea is a relatively simple, avascular organ that offers an exceptional model to better understand the pathophysiology of the fibrosis response. Injury and defective regeneration of the epithelial basement membrane (EBM) or the endothelial Descemet's basement membrane (DBM) triggers the development of myofibroblasts from resident corneal fibroblasts and bone marrow-derived blood borne fibrocytes due to the increased entry of TGF beta-1/-2 into the stroma from the epithelium and tears or residual corneal endothelium and aqueous humor. The myofibroblasts, and disordered extracellular matrix these cells produce, persist until the source of injury is removed, the EBM and/or DBM are regenerated, or replaced surgically, resulting in decreased stromal TGF beta requisite for myofibroblast survival. A similar BM injury-related pathophysiology can underly the development of fibrosis in other organs such as skin and lung. The normal liver does not contain traditional BMs but develops sinusoidal endothelial BMs in many fibrotic diseases and models. However, normal hepatic stellate cells produce collagen type IV and perlecan that can modulate TGF beta localization and cognate receptor binding in the space of Dissé. BM-related fibrosis is deserving of more investigation in all organs.

The role of basement membranes in cardiac biology and disease.

Basement membranes (BMs) are highly specialised extracellular matrix (ECM) structures that within the heart underlie endothelial cells (ECs) and surround cardiomyocytes and vascular smooth muscle cells. They generate a dynamic and structurally supportive environment throughout cardiac development and maturation by providing physical anchorage to the underlying interstitium, structural support to the tissue, and by influencing cell behaviour and signalling. While this provides a strong link between BM dysfunction and cardiac disease, the role of the BM in cardiac biology remains under-researched and our understanding regarding the mechanistic interplay between BM defects and their morphological and functional consequences remain important knowledge-gaps. In this review, we bring together emerging understanding of BM defects within the heart including in common cardiovascular pathologies such as contractile dysfunction and highlight some key questions that are now ready to be addressed.

Role of albumin on endothelial basement membrane and hemostasis in a rat model of hemorrhagic shock.

BACKGROUND: We sought to determine the extent of loss of endothelial basement membrane (BM), leukocyte recruitment, and changes in coagulation after hemorrhagic shock, followed by limited-volume resuscitation (LVR) with 5% albumin (ALB). METHODS: Anesthetized rats were bled 40% of blood volume and assigned to treatment groups: untreated (n = 6), LVR with normal saline (NS; n = 8), or LVR with ALB (n = 8). Sham rats (n = 6) underwent all procedures except hemorrhage or resuscitation. Blood samples were assayed for active proteases, such as metalloproteinase 9 (MMP-9) and a disintegrin and metalloproteinase 10 (ADAM-10), BM-type heparan sulfate proteoglycan (perlecan), cell count, and coagulation function. Leukocyte transmigration was used to estimate the net efficiency of leukocyte recruitment in cremaster venules. RESULTS: Hemorrhage significantly lowered red cell count, but white cell and platelet counts did not change (vs. sham). Ionized calcium in plasma was significantly reduced in untreated and remained so after NS. In contrast, ionized calcium was normalized after ALB. Plasma expansion after NS and ALB further reduced leukocyte and platelet counts. Metalloproteinase 9, ADAM-10, and perlecan were significantly higher in untreated rats (vs. sham). Albumin normalized MMP-9, ADAM-10, and perlecan levels, while NS further increased MMP-9, ADAM-10, and perlecan (vs. sham). Transmigrated leukocytes doubled in the untreated group and remained elevated after NS (vs. sham) but normalized after ALB. Albumin reduced every stage of the leukocyte recruitment process to sham levels. CONCLUSION: Despite similar plasma expansion, NS weakened platelet function contrary to ALB. Plasma expansion with ALB resulted in restoration of BM integrity and attenuation of leukocyte recruitment to tissues, in contrast to NS. Albumin plays a critical role in restoring BM integrity, attenuating leukocyte recruitment to tissues, and optimizing hemostasis by increasing ionized calcium in plasma.

Upregulation of VCAM-1 in lymphatic collectors supports dendritic cell entry and rapid migration to lymph nodes in inflammation.

Dendritic cell (DC) migration to draining lymph nodes (dLNs) is a slow process that is believed to begin with DCs approaching and entering into afferent lymphatic capillaries. From capillaries, DCs slowly crawl into lymphatic collectors, where lymph flow induced by collector contraction supports DC detachment and thereafter rapid, passive transport to dLNs. Performing a transcriptomics analysis of dermal endothelial cells, we found that inflammation induces the degradation of the basement membrane (BM) surrounding lymphatic collectors and preferential up-regulation of the DC trafficking molecule VCAM-1 in collectors. In crawl-in experiments performed in ear skin explants, DCs entered collectors in a CCR7- and ß1 integrin-dependent manner. In vivo, loss of ß1-integrins in DCs or of VCAM-1 in lymphatic collectors had the greatest impact on DC migration to dLNs at early time points when migration kinetics favor the accumulation of rapidly migrating collector DCs rather than slower capillary DCs. Taken together, our findings identify collector entry as a critical mechanism enabling rapid DC migration to dLNs in inflammation.

FUNCTIONAL AND MORPHOLOGICAL OUTCOMES OF THE INVERTED INTERNAL LIMITING MEMBRANE FLAP TECHNIQUE IN SMALL-SIZED AND MEDIUM-SIZED MACULAR HOLES <400 µm.

PURPOSE: To assess the effect of an internal limiting membrane flap (IF) in macular hole surgery on the best-corrected visual acuity (BCVA) and integrity of the ellipsoid zone (EZ) and external limiting membrane. METHODS: Patients were included who had successful surgery for macular hole <400 µm with or without an IF. Main outcome measures were BCVA and restoration of the external limiting membrane and EZ at 12 months. RESULTS: Sixty patients were included, 36 with conventional peeling and 24 with an IF. The best-corrected visual acuity improved from 0.74 (±0.30) logarithm of the minimum angle of resolution (20/110 Snellen) to 0.26 (±0.20) (20/36 Snellen) in patients without and from 0.77 (±0.32) logarithm of the minimum angle of resolution (20/118 Snellen) to 0.18 (±0.12) (20/30 Snellen) in patients with an IF, respectively. There was no difference in the integrity of the EZ and external limiting membrane in patients with or without an IF at either 3 (P = 0.58, P = 0.20), 6 (P = 0.81, P = 0.10), or 12 months (P = 0.60, P = 0.20) or in the BCVA at 3 (P = 0.24), 6 (P = 0.18) and 12 months (P = 0.11). In the multivariable model, only preoperative BCVA (P < 0.01), EZ integrity (P = 0.001), and age (P < 0.01) were associated with the post-operative BCVA. CONCLUSION: In patients undergoing surgery for macular hole <400 µm, the use of an IF did not affect the BCVA or the integrity of the EZ and external limiting membrane.

Spontaneous Resolution of Foveal Sub-internal Limiting Membrane Hemorrhage with Excellent Visual and Anatomical Outcome in a Patient with Acute Myeloid Leukemia.

Sub-internal limiting membrane (sub-ILM) hemorrhage is a distinct type of retinal hemorrhage in which the blood accumulates between ILM and nerve fiber layer. Little is known about visual prognosis as well as ideal management of foveal sub-ILM hemorrhage in patients with acute leukemia. Herein, we presented a case of acute myeloid leukemia with foveal sub-ILM hemorrhage. Observation alone resulted in complete resolution of hemorrhage with good visual and anatomical outcome.

Bilateral and multiple sub-internal limiting membrane hemorrhages in a familial retinal arteriolar tortuosity patient by Valsalva-like mechanism: an observational case report.

BACKGROUND: Bilateral and multiple Valsalva-related sub-internal limiting membrane (ILM) hemorrhages in a familial retinal arteriolar tortuosity (FRAT) patient is rare, and we treated this patient by both observation and Neodymium yttrium aluminum garnet (Nd: YAG) laser membranotomy methods. CASE PRESENTATION: A 13-year-old female student presented with sudden visual loss and central scotoma in both eyes after running 800 m at the school gym. The examination revealed six sub-ILM hemorrhages with the biggest hemorrhage measuring approximately 1.5-disc diameters (DD) in the right eye and two sub-ILM hemorrhages with the biggest one measuring 5.5 DD in the left eye. The patient was diagnosed as having Valsalva retinopathy associated with FRAT. Nd: YAG laser membranotomy was performed at the biggest hemorrhages and the rest hemorrhages were treated with observation in both eyes. The visual acuity recovered to 20/16 in the right eye and 20/20 in the left eye. Epiretinal membrane (ERM) formation was observed in the left eye. CONCLUSIONS: Nd: YAG laser could be considered for treating premacular hemorrhage in FRAT patient especially when a quick vision recovery was needed. This is the first reported case of a FRAT patient suffering from bilateral and multiple Valsalva-related sub-ILM hemorrhages which were treated by both observation and Nd: YAG laser treatment.

Basement Membrane Remodeling Controls Endothelial Function in Idiopathic Pulmonary Arterial Hypertension.

The extracellular matrix (ECM) increasingly emerges as an active driver in several diseases, including idiopathic pulmonary arterial hypertension (IPAH). The basement membrane (BM) is a specialized class of ECM proteins. In pulmonary arteries, the BM is in close contact and direct proximity to vascular cells, including endothelial cells. So far, the role of the BM has remained underinvestigated in IPAH. Here, we aimed to shed light on the involvement of the BM in IPAH, by addressing its structure, composition, and function. On an ultrastructural level, we observed a marked increase in BM thickness in IPAH pulmonary vessels. BM composition was distinct in small and large vessels and altered in IPAH. Proteoglycans were mostly responsible for distinction between smaller and larger vessels, whereas BM collagens and laminins were more abundantly expressed in IPAH. Type IV collagen and laminin both strengthened endothelial barrier integrity. However, only type IV collagen concentration dependently increased cell adhesion of both donor and IPAH-derived pulmonary arterial endothelial cells (PAECs) and induced nuclear translocation of mechanosensitive transcriptional coactivator of the hippo pathway YAP (Yes-activated protein). On the other hand, laminin caused cytoplasmic retention of YAP in IPAH PAECs. Accordingly, silencing of COL4A5 and LAMC1, respectively, differentially affected tight junction formation and barrier integrity in both donor and IPAH PAECs. Collectively, our results highlight the importance of a well-maintained BM homeostasis. By linking changes in BM structure and composition to altered endothelial cell function, we here suggest an active involvement of the BM in IPAH pathogenesis.

Sub-perfluoro-n-octane injection of ocular viscoelastic device assisted inverted internal limiting membrane flap for macular hole retinal detachment surgery: a novel technique.

BACKGROUND: To evaluate the feasibility of a surgical technique using a sub-perfluoro-n-octane (PFO) injection of ocular viscoelastic device (OVD) to stabilize inverted internal limiting membrane (ILM) flap for the treatment of macular hole retinal detachment (MHRD). METHODS: This study was a retrospective, consecutive, interventional case series. Patients who underwent MHRD surgery with sub-PFO injection of OVD to stabilize inverted ILM flap onto the macular hole (MH) were reviewed. The color fundus and optical coherence tomography (OCT) images were collected and evaluated. The best-corrected visual acuity (BCVA) before and after surgery were compared as the functional outcome. RESULTS: The study included 8 eyes of 8 consecutive patients (mean age: 61.8 ± 7.1 years; mean follow-up period: 9.0 ± 2.5 months). All eyes (100%) achieved successful MH closure; 7 eyes (87.5%) demonstrated complete retinal reattachment, and 1 eye (12.5%) had minimal residual subretinal fluid parafoveally. Of the 8 patients, 7 patients (87.5%) had achieved improvement in BCVA after the primary surgery, whereas 1 eye remained stable. The average BCVA before and after the surgery at the last visit improved from 20/843 (1.63 ± 0.48 logMAR) to 20/200 (1.00 ± 0.39 logMAR) (P = 0.016). Anatomically, near-normal foveal contour was noted in five (62.5%) eyes at the final follow-up. CONCLUSIONS: The use of sub-PFO injection of OVD in MHRD surgery could stabilize inverted ILM flaps, achieve good anatomical results and improve postoperative BCVA.

Comparative efficacy evaluation of inverted internal limiting membrane flap technique and internal limiting membrane peeling in large macular holes: a systematic review and meta-analysis.

BACKGROUND: The purpose of this study was to compare the anatomical and visual outcomes of inverted internal limiting membrane (ILM) flap technique and internal limiting membrane peeling in large macular holes (MH). METHODS: Related studies were reviewed by searching electronic databases of Pubmed, Embase, Cochrane Library. We searched for articles that compared inverted ILM flap technique with ILM peeling for large MH (> 400 µm). Double-arm meta-analysis was performed for the primary end point that was the rate of MH closure, and the secondary end point was postoperative visual acuity (VA). Heterogeneity, publication bias, sensitivity analysis and subgroup analysis were conducted to guarantee the statistical power. RESULTS: This review included eight studies involving 593 eyes, 4 randomized control trials and 4 retrospective studies. After sensitivity analysis for eliminating the heterogeneity of primary outcome, the pooled data showed the rate of MH closure with inverted ILM flap technique group was statistically significantly higher than ILM peeling group (odds ratio (OR) = 3.95, 95% confidence interval (CI) = 1.89 to 8.27; P = 0.0003). At the follow-up duration of 3 months, postoperative VA was significantly better in the group of inverted ILM flap than ILM peeling (mean difference (MD) = - 0.16, 95% CI = - 0.23 to 0.09; P < 0.00001). However, there was no difference in visual outcomes between the two groups of different surgical treatments at relatively long-term follow-up over 6 months (MD = 0.01, 95% CI = - 0.12 to 0.15; P = 0.86). CONCLUSION: Vitrectomy with inverted ILM flap technique had a better anatomical outcome than ILM peeling. Flap technique also had a signifcant visual gain in the short term, but the limitations in visual recovery at a longer follow-up was found.

FOVEAL SPARING INTERNAL LIMITING MEMBRANE PEELING FOR IDIOPATHIC MACULAR HOLES: EFFECTS ON ANATOMICAL RESTORATION OF THE FOVEA AND VISUAL FUNCTION.

PURPOSE: Muller cells seem to be important in maintaining foveal morphology through connections between their foot processes and the internal limiting membrane (ILM). Internal limiting membrane peeling causes Muller cell trauma. We hypothesized that leaving a rim of unpeeled ILM around idiopathic macular holes undergoing vitrectomy surgery would improve postoperative foveal morphology and vision. METHODS: Prospective pilot study of fovea-sparing ILM peeling in a consecutive cohort of patients with macular holes over a 12-month period. Spectral-domain optical coherence tomography and Early Treatment Diabetic Retinopathy Study letters best-corrected visual acuity were assessed preoperatively and postoperatively, and foveal morphology and metamorphopsia postoperatively. The foveal sparing group was compared with a second consecutive cohort who received standard ILM peeling (control group). RESULTS: Thirty-four eyes of 34 patients were included in each group. Groups showed no significant preoperative differences. 34/34 holes were successfully closed with surgery in the foveal sparing group and 32/34 in the control group. The foveal sparing group showed better postoperative best-corrected visual acuity (67.7 vs. 63.8, P = 0.003) and best-corrected visual acuity improvement (25.1 vs. 20.2, P = 0.03). The foveal sparing group demonstrated thicker minimum foveal thickness (211 vs. 173 µm, P = 0.002) and less steep foveal depression (158 vs. 149, P = 0.002). CONCLUSION: Preserving nonpeeled ILM around macular holes resulted in a high closure rate, improved foveal morphology, and better postoperative best-corrected visual acuity. An appropriately powered randomized controlled study is warranted.

Fovea-sparing internal limiting membrane peeling versus complete internal limiting membrane peeling for myopic traction maculopathy.

PURPOSE: To compare the outcomes of vitrectomy with fovea-sparing internal limiting membrane peeling (FSIP) and complete internal limiting membrane peeling (ILMP) for myopic traction maculopathy (MTM). STUDY DESIGN: A retrospective, observational study. PATIENTS AND METHODS: In this study, we included 22 eyes of 21 consecutive patients who underwent vitrectomy with FSIP or ILMP for MTM and were monitored for at least 6 months. Eleven eyes were treated with FSIP, and 11, with ILMP. RESULTS: With FSIP, the postoperative best-corrected visual acuity (BCVA) significantly improved from 0.61 (20/82) to 0.34 (20/44; P = .009) logarithm of the minimum angle of resolution (logMAR) units. With ILMP, the postoperative BCVA improved from 0.65 (20/89) to 0.52 (20/66) logMAR units, but was not significant (P = .106). The postoperative final central foveal thickness (CFT) reduced significantly after FSIP (from 557.6 to 128.8 µm, P = .003) and ILMP (from 547.3 to 130.3 µm, P = .008). The postoperative incidence of a macular hole was 0% (0/11 eyes) with FSIP and 27.3% (3/11 eyes) with ILMP. All patients with a macular hole had foveal detachment in association with a thin fovea preoperatively. With ILMP, postoperative BCVA with a macular hole worsened by -3.5 letters; in contrast, postoperative BCVA without a macular hole improved by +10.5 letters. With FSIP, postoperative BCVA without a macular hole significantly improved by +13.5 letters (P = .009). CONCLUSIONS: FSIP resulted in significant improvement in MTM and prevented postoperative macular hole development.

Basement membrane and blood-brain barrier.

The blood-brain barrier (BBB) is a highly complex and dynamic structure, mainly composed of brain microvascular endothelial cells, pericytes, astrocytes and the basement membrane (BM). The vast majority of BBB research focuses on its cellular constituents. Its non-cellular component, the BM, on the other hand, is largely understudied due to its intrinsic complexity and the lack of research tools. In this review, we focus on the role of the BM in BBB integrity. We first briefly introduce the biochemical composition and structure of the BM. Next, the biological functions of major components of the BM in BBB formation and maintenance are discussed. Our goal is to provide a concise overview on how the BM contributes to BBB integrity.

Neutrophil elastase plays a non-redundant role in remodeling the venular basement membrane and neutrophil diapedesis post-ischemia/reperfusion injury.

Ischemia/reperfusion (I/R) injury is a severe inflammatory insult associated with numerous pathologies, such as myocardial infarction, stroke and acute kidney injury. I/R injury is characterized by a rapid influx of activated neutrophils secreting toxic free radical species and degrading enzymes that can irreversibly damage the tissue, thus impairing organ functions. Significant efforts have been invested in identifying therapeutic targets to suppress neutrophil recruitment and activation post-I/R injury. In this context, pharmacological targeting of neutrophil elastase (NE) has shown promising anti-inflammatory efficacy in a number of experimental and clinical settings of I/R injury and is considered a plausible clinical strategy for organ care. However, the mechanisms of action of NE, and hence its inhibitors, in this process are not fully understood. Here we conducted a comprehensive analysis of the impact of NE genetic deletion on neutrophil infiltration in four murine models of I/R injury as induced in the heart, kidneys, intestine and cremaster muscle. In all models, neutrophil migration into ischemic regions was significantly suppressed in NE-/- mice as compared with wild-type controls. Analysis of inflamed cremaster muscle and mesenteric microvessels by intravital and confocal microscopy revealed a selective entrapment of neutrophils within venular walls, most notably at the level of the venular basement membrane (BM) following NE deletion/pharmacological blockade. This effect was associated with the suppression of NE-mediated remodeling of the low matrix protein expressing regions within the venular BM used by transmigrating neutrophils as exit portals. Furthermore, whilst NE deficiency led to reduced neutrophil activation and vascular leakage, levels of monocytes and prohealing M2 macrophages were reduced in tissues of NE-/- mice subjected to I/R. Collectively our results identify a vital and non-redundant role for NE in supporting neutrophil breaching of the venular BM post-I/R injury but also suggest a protective role for NE in promoting tissue repair. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Morphologic, Biomechanical, and Compositional Features of the Internal Limiting Membrane in Pathologic Myopic Foveoschisis.

Purpose: To investigate alterations in the morphologic, compositional, and biomechanical properties of the internal limiting membrane (ILM) in pathologic myopic foveoschisis (MF) eyes. Methods: ILM specimens were peeled from 61 eyes with MF and 56 eyes with stage III/IV idiopathic macular hole (IMH) as a control. Samples were analyzed for transmission electron microscopy (TEM), scanning electron microscopy, immunofluorescence, Western blotting, and atomic force microscopy. ILM characteristics were compared between the two groups. Results: TEM findings revealed that thickness of the MF ILMs significantly decreased compared with that of IMH ILMs (0.753 ± 0.215 vs. 1.894 ± 0.247 µm; P < 0.0001). The vitreal side stiffness of the MF ILMs was markedly higher than that of the IMH ILMs (3.520 ± 0.803 vs. 0.879 ± 0.230 MPa, P < 0.0001). Comparing with the IMH group, collagen IV exhibited decreased concentration and different immunofluorescence distribution in ILMs of MF eyes, so also protein α3 (IV), α4 (IV), and α5 (IV). The immunofluorescence staining results showed that astrocytes were observed in none of the IMH eyes and in 12 of 16 MF eyes (75%, P < 0.0001). Conclusions: These alterations in the MF ILMs appear to be associated with Müller cell and astrocyte reactive gliosis. The present findings contribute to a more in-depth understanding of the pathogenesis of MF.

Randall's plaque in stone formers originates in ascending thin limbs.

Randall's plaque, an attachment site over which calcium oxalate stones form, begins in the basement membranes of thin limbs of the loop of Henle. The mechanism of its formation is unknown. Possibly, enhanced delivery of calcium out of the proximal tubule, found in many stone formers, increases reabsorption of calcium from the thick ascending limb into the interstitium around descending vasa recta, which convey that calcium into the deep medulla, and raises supersaturations near thin limbs ("vas washdown"). According to this hypothesis, plaque should form preferentially on ascending thin limbs, which do not reabsorb water. We stained serial sections of papillary biopsies from stone-forming patients for aquaporin 1 (which is found in the descending thin limb) and the kidney-specific chloride channel ClC-Ka (which is found in the ascending thin limb). Plaque (which is detected using Yasue stain) colocalized with ClC-Ka, but not with aquaporin 1 (χ2 = 464, P < 0.001). We conclude that plaque forms preferentially in the basement membranes of ascending thin limbs, fulfilling a critical prediction of the vas washdown theory of plaque pathogenesis. The clinical implication is that treatments such as a low-sodium diet or thiazide diuretics that raise proximal tubule calcium reabsorption may reduce formation of plaque as well as calcium kidney stones.

Neurodegeneration in diabetic retinopathy: does it really matter?

The concept of diabetic retinopathy as a microvascular disease has evolved, in that it is now considered a more complex diabetic complication in which neurodegeneration plays a significant role. In this article we provide a critical overview of the role of microvascular abnormalities and neurodegeneration in the pathogenesis of diabetic retinopathy. A special emphasis is placed on the pathophysiology of the neurovascular unit (NVU), including the contributions of microvascular and neural elements. The potential mechanisms linking retinal neurodegeneration and early microvascular impairment, and the effects of neuroprotective drugs are summarised. Additionally, we discuss how the assessment of retinal neurodegeneration could be an important index of cognitive status, thus helping to identify individuals at risk of dementia, which will impact on current procedures for diabetes management. We conclude that glial, neural and microvascular dysfunction are interdependent and essential for the development of diabetic retinopathy. Despite this intricate relationship, retinal neurodegeneration is a critical endpoint and neuroprotection, itself, can be considered a therapeutic target, independently of its potential impact on microvascular disease. In addition, interventional studies targeting pathogenic pathways that impact the NVU are needed. Findings from these studies will be crucial, not only for increasing our understanding of diabetic retinopathy, but also to help to implement a timely and efficient personalised medicine approach for treating this diabetic complication.