Basement membrane stiffening promotes retinal endothelial activation associated with diabetes.

Endothelial activation is a hallmark of the high-glucose (HG)-induced retinal inflammation associated with diabetic retinopathy (DR). However, precisely how HG induces retinal endothelial activation is not fully understood. We hypothesized that HG-induced up-regulation of lysyl oxidase (LOX), a collagen-cross-linking enzyme, in retinal capillary endothelial cells (ECs) enhances subendothelial basement membrane (BM) stiffness, which, in turn, promotes retinal EC activation. Diabetic C57BL/6 mice exhibiting a 70 and 50% increase in retinal intercellular adhesion molecule (ICAM)-1 expression and leukocyte accumulation, respectively, demonstrated a 2-fold increase in the levels of BM collagen IV and LOX, key determinants of capillary BM stiffness. Using atomic force microscopy, we confirmed that HG significantly enhances LOX-dependent subendothelial matrix stiffness in vitro, which correlated with an ∼2.5-fold increase in endothelial ICAM-1 expression, a 4-fold greater monocyte-EC adhesion, and an ∼2-fold alteration in endothelial NO (decrease) and NF-κB activation (increase). Inhibition of LOX-dependent subendothelial matrix stiffening alone suppressed HG-induced retinal EC activation. Finally, using synthetic matrices of tunable stiffness, we demonstrated that subendothelial matrix stiffening is necessary and sufficient to promote EC activation. These findings implicate BM stiffening as a critical determinant of HG-induced retinal EC activation and provide a rationale for examining BM stiffness and underlying mechanotransduction pathways as therapeutic targets for diabetic retinopathy.

Senescence Increases Choroidal Endothelial Stiffness and Susceptibility to Complement Injury: Implications for Choriocapillaris Loss in AMD.

PURPOSE: Age-related macular degeneration (AMD) commonly causes blindness in the elderly. Yet, it is untreatable in the large fraction of all AMD patients that develop the early dry form. Dry AMD is marked by the deposition of membrane attack complex (MAC) on choriocapillaris (CC), which is implicated in CC degeneration and subsequent atrophy of overlying retinal pigment epithelium. Since MAC is also found on the CC of young eyes, here we investigated whether and how aging increases choroidal endothelial susceptibility to MAC injury. METHODS: Monkey chorioretinal endothelial cells (ECs, RF/6A) were cultured to high passages (>P60) to achieve replicative senescence. We treated ECs with complement-competent human serum to promote MAC deposition and injury, which were assessed by flow cytometry and trypan blue exclusion assay, respectively. Stiffness of EC was measured by atomic force microscopy indentation while Rho GTPase activity was quantified by Rho G-LISA assay. RESULTS: Our findings reveal that senescent ECs are significantly stiffer than their normal counterparts, which correlates with higher cytoskeletal Rho activity in these cells and their greater susceptibility to complement (MAC) injury. Importantly, inhibition of Rho activity in senescent ECs significantly reduced cell stiffness and MAC-induced lysis. CONCLUSIONS: By revealing an important role of senescence-associated choroidal EC stiffening in complement injury, these findings implicate CC stiffening as an important determinant of age-related CC atrophy seen in dry AMD. Future studies are needed to validate these findings in appropriate animal models so new therapeutic targets can be identified for treatment of dry AMD.

Matrix stiffness exerts biphasic control over monocyte-endothelial adhesion via Rho-mediated ICAM-1 clustering.

Leukocyte-endothelial adhesion is a critical early step in chronic vascular inflammation associated with diabetes, emphysema, and aging. Importantly, these conditions are also marked by abnormal subendothelial matrix crosslinking (stiffness). Yet, whether and how abnormal matrix stiffness contributes to leukocyte-endothelial adhesion remains poorly understood. Using a co-culture of human monocytic cells and human microvascular endothelial cells (ECs) grown on matrices of tunable stiffness, we demonstrate that matrix stiffness exerts biphasic control over monocyte-EC adhesion, with both matrix softening and stiffening eliciting a two-fold increase in this adhesive interaction. This preferential endothelial adhesivity on softer and stiffer matrices was consistent with a significant increase in α-actinin-4-associated endothelial ICAM-1 clustering, a key determinant of monocyte-EC adhesion. Further, the enhanced ICAM-1 clustering on soft and stiff matrices correlated strongly with an increase in Rho activity and ROCK2 expression. Importantly, inhibition of Rho/ROCK activity blocked the effects of abnormal matrix stiffness on ICAM-1 clustering and monocyte-EC adhesion. Thus, these findings implicate matrix stiffness-dependent ICAM-1 clustering as an important regulator of vascular inflammation and provide the rationale for closely examining mechanotransduction pathways as new molecular targets for anti-inflammatory therapy.

Nanoliposomal Nitroglycerin Exerts Potent Anti-Inflammatory Effects.

Nitroglycerin (NTG) markedly enhances nitric oxide (NO) bioavailability. However, its ability to mimic the anti-inflammatory properties of NO remains unknown. Here, we examined whether NTG can suppress endothelial cell (EC) activation during inflammation and developed NTG nanoformulation to simultaneously amplify its anti-inflammatory effects and ameliorate adverse effects associated with high-dose NTG administration. Our findings reveal that NTG significantly inhibits human U937 cell adhesion to NO-deficient human microvascular ECs in vitro through an increase in endothelial NO and decrease in endothelial ICAM-1 clustering, as determined by NO analyzer, microfluorimetry, and immunofluorescence staining. Nanoliposomal NTG (NTG-NL) was formulated by encapsulating NTG within unilamellar lipid vesicles (DPhPC, POPC, Cholesterol, DHPE-Texas Red at molar ratio of 6:2:2:0.2) that were ~155 nm in diameter and readily uptaken by ECs, as determined by dynamic light scattering and quantitative fluorescence microscopy, respectively. More importantly, NTG-NL produced a 70-fold increase in NTG therapeutic efficacy when compared with free NTG while preventing excessive mitochondrial superoxide production associated with high NTG doses. Thus, these findings, which are the first to reveal the superior therapeutic effects of an NTG nanoformulation, provide the rationale for their detailed investigation for potentially superior vascular normalization therapies.

Aberrant cell and basement membrane architecture contribute to sidestream smoke-induced choroidal endothelial dysfunction.

PURPOSE: Environmental tobacco smoke (ETS) is widely regarded as a major modifiable risk factor for age-related macular degeneration (AMD). Yet, precisely how it exerts its pathologic effects is poorly understood. Since early-stage AMD is characterized by choroidal capillary loss, this study examined the effect of sidestream smoke (SS), the major component of ETS, on the viability of choroidal endothelial cells (EC), with an emphasis on the role of aberrant cell and basement membrane (BM) architecture in mediating SS-induced response. METHODS: Chorioretinal ECs (RF/6A) were treated with SS, and cell viability and architecture were analyzed by colorimetric assay and actin cytoskeletal organization, respectively. The structure of RF/6A EC-secreted BM was examined by immunofluorescence for collagen IV and immunoblotting for lysyl oxidase (LOX), a collagen-crosslinking enzyme. Finally, fresh RF/6A ECs were cultured on decellularized SS-treated BM to evaluate its active role in EC dysfunction. RESULTS: The RF/6A EC viability decreased progressively with increasing SS dose, which correlated strongly with a significant decline in actin cytoskeleton-dependent EC spreading. Sidestream smoke also caused marked disruption of the RF/6A EC-secreted BM that was accompanied by suppression of LOX expression. Further, fresh, non-SS-treated RF/6A ECs exhibited a significant loss in viability and actin cytoskeletal organization when cultured on SS-treated corrupt BM. CONCLUSIONS: These findings indicate that aberrant physical cues in the form of EC and BM architecture likely have an important role in choriocapillaris dysfunction seen in SS-associated early AMD and implicate choroidal BM as a potential target for AMD management strategies.