Endothelial Cell Morphology Research Articles

Micro-optical coherence tomography for high-resolution morphologic imaging of cellular and nerval corneal micro-structures.

We demonstrate the highest resolution (1.5×1.5×1 µm) micrometer optical coherence tomography (µOCT) imaging of the morphologic micro-structure of excised swine and non-human primate corneas. Besides epithelial, stromal, and endothelial cell morphology, this report focuses on investigating the most peripheral corneal nerve fibers, the nerve fibers of the subbasal plexus (SBP). Alterations of SBP nerve density and composition are reportedly linked to major neurologic disorders, such as diabetic neuropathy, potentially indicating earliest onsets of denervation. Here, the fine, hyperreflective, epithelial nerve structures located just above Bowman's membrane, are i) visualized using our µOCT prototype, ii) validated by comparison to fluorescence confocal microscopy (including selective immunohistochemical staining), and iii) segmented using state-of-the-art image processing. Here, we also introduce polarization sensitive (PS) µOCT imaging, demonstrating, to the best of our knowledge, the highest resolution corneal PS-OCT scans reported to date.

Apelin signaling drives vascular endothelial cells toward a pro-angiogenic state.

To form new blood vessels (angiogenesis), endothelial cells (ECs) must be activated and acquire highly migratory and proliferative phenotypes. However, the molecular mechanisms that govern these processes are incompletely understood. Here, we show that Apelin signaling functions to drive ECs into such an angiogenic state. Zebrafish lacking Apelin signaling exhibit defects in endothelial tip cell morphology and sprouting. Using transplantation experiments, we find that in mosaic vessels, wild-type ECs leave the dorsal aorta (DA) and form new vessels while neighboring ECs defective in Apelin signaling remain in the DA. Mechanistically, Apelin signaling enhances glycolytic activity in ECs at least in part by increasing levels of the growth-promoting transcription factor c-Myc. Moreover, APELIN expression is regulated by Notch signaling in human ECs, and its function is required for the hypersprouting phenotype in Delta-like 4 (Dll4) knockdown zebrafish embryos. These data provide new insights into fundamental principles of blood vessel formation and Apelin signaling, enabling a better understanding of vascular growth in health and disease.

Stem Cell Repair of the Microvascular Damage in Stroke.

Stroke is a life-threatening disease that leads to mortality, with survivors subjected to long-term disability. Microvascular damage is implicated as a key pathological feature, as well as a therapeutic target for stroke. In this review, we present evidence detailing subacute diaschisis in a focal ischemic stroke rat model with a focus on blood–brain barrier (BBB) integrity and related pathogenic processes in contralateral brain areas. Additionally, we discuss BBB competence in chronic diaschisis in a similar rat stroke model, highlighting the pathological changes in contralateral brain areas that indicate progressive morphological brain disturbances overtime after stroke onset. With diaschisis closely approximating stroke onset and progression, it stands as a treatment of interest for stroke. Indeed, the use of stem cell transplantation for the repair of microvascular damage has been investigated, demonstrating that bone marrow stem cells intravenously transplanted into rats 48 h post-stroke survive and integrate into the microvasculature. Ultrastructural analysis of transplanted stroke brains reveals that microvessels display a near-normal morphology of endothelial cells and their mitochondria. Cell-based therapeutics represent a new mechanism in BBB and microvascular repair for stroke.

Effect of microalbuminuria on corneal endothelium in patients with diabetes without retinopathy

BackgroundThe aim of this study was to investigate the effect of microalbuminuria on corneal endothelium in patients with diabetes without retinopathy.MethodsThis cross‐sectional study comprised 100 patients with type 2 diabetes mellitus (DM) without diabetic retinopathy and 92 control subjects without diabetes. Forty‐five patients had microalbuminuria and 55 subjects were microalbuminuria negative. Endothelial measurements were obtained using specular microscopy. Endothelial cell density, average area, co‐efficient of variation, maximum area, minimum area, hexagonality and corneal thickness were compared between the groups on the basis of microalbuminuria, DM duration and medication, HbA1c, body mass index, serum lipid and protein profiles, hypertension and hyperlipidaemia diagnosis.ResultsThere were no significant differences in age, gender, endothelial cell density, average area, co‐efficients of variation, maximum area, minimum area, hexagonality or corneal thickness among the microalbuminuria positive, microalbuminuria negative and control groups (p > 0.05). However, microalbuminuria positive patients had lower high‐density lipoprotein levels than the microalbuminuria negative patients (p = 0.042). DM and control groups showed similar endothelial measurements (p > 0.05). Patients with a HbA1c > 7 per cent (53 mmol/mol) had lower hexagonality value (p = 0.039) than in the subjects with a HbA1c ≤ 7 per cent. No significant differences were found in endothelial parameters when groups were compared based on DM duration, medication and co‐morbidities.ConclusionsMicroalbuminuria positive and negative patients with DM without retinopathy seem to have similar corneal endothelial measurements with controls. However, patients with a HbA1c > 7 per cent should be monitored for deterioration in corneal endothelial cell morphology even without diabetic retinopathy, which might be critical prior to anterior segment surgery.

Endothelial to mesenchymal transition: a precursor to post-COVID-19 interstitial pulmonary fibrosis and vascular obliteration?

Endothelial to mesenchymal transition (EndMT) could lead to post-COVID-19 pulmonary fibrosis and vascular remodelling https://bit.ly/2QqSKxT

Bicuspid Aortic Valve and Endothelial Dysfunction: Current Evidence and Potential Therapeutic Targets.

Bicuspid aortic valve (BAV), the most frequent congenital heart malformation, is characterized by the presence of a two-leaflet aortic valve instead of a three-leaflet one. BAV disease progression is associated with valvular dysfunction (in the form of stenosis or regurgitation) and aortopathy, which can lead to aneurysm and aortic dissection. This morphological abnormality modifies valve dynamics and promotes eccentric blood flow, which gives rise to alterations of the flow pattern and wall shear stress (WSS) of the ascending aorta. Recently, evidence of endothelial dysfunction (ED) in BAV disease has emerged. Different studies have addressed a reduced endothelial functionality by analyzing various molecular biomarkers and cellular parameters in BAV patients. Some authors have found impaired functionality of circulating endothelial progenitors in these patients, associating it with valvular dysfunction and aortic dilation. Others focused on systemic endothelial function by measuring artery flow-mediated dilation (FMD), showing a reduced FMD in BAV individuals. Novel biomarkers like increased endothelial microparticles (EMP), which are related to ED, have also been discovered in BAV patients. Finally, latest studies indicate that in BAV, endothelial-to-mesenchymal transition (EndoMT) may also be de-regulated, which could be caused by genetic, hemodynamic alterations, or both. Different hypothesis about the pathology of ED in BAV are nowadays being debated. Some authors blamed this impaired functionality just on genetic abnormalities, which could lead to a pathological aorta. Nevertheless, thanks to the development of new and high-resolution imaging techniques like 4D flow MRI, hemodynamics has gained great attention. Based on latest studies, alterations in blood flow seem to cause proper modification of the endothelial cells (ECs) function and morphology. It also seems to be associated with aortic dilation and decreased vasodilators expression, like nitric oxide (NO). Although nowadays ED in BAV has been reported by many, it is not clear which its main cause may be. Comprehending the pathways that promote ED and its relevance in BAV could help further understand and maybe prevent the serious consequences of this disease. This review will discuss the ED present in BAV, focusing on the latest evidence, biomarkers for ED and potential therapeutic targets (Figure 1).

The influence of preparation and storage time on endothelial cells in Quarter-Descemet membrane endothelial keratoplasty (Quarter-DMEK) grafts in vitro.

Quarter-Descemet membrane endothelial keratoplasty (Quarter-DMEK) has been introduced as a modification of the standard DMEK technique to increase the pool of endothelial grafts. In this study, we evaluated in vitro changes in endothelial cell distribution, viability and morphology of Quarter-DMEK grafts when stored in organ-culture medium. Quarter-DMEK grafts were prepared from 5 corneas and stored in organ-culture medium for 4, 7 and 11days. Endothelial cell re-distribution was investigated by light microscopy, cell viability by a Calcein-AM assay, and expression of endothelial and non-endothelial markers by immunohistochemistry. Three standard DMEK-grafts were used as controls. After preparation, all Quarter-DMEK grafts showed a band with no viable endothelial cells along the radial cut graft edges [average width 190 (± 20)µm]. Endothelial cell density in the central graft area decreased by 12%, 23% and 26% after 4, 7, and 11days of storage, respectively. At the same time, empty bands along the cut edges were re-populated and some cells migrated to the stromal side of the Descemet membrane (DM). These cells showed an altered phenotype, as indicated by expression of migration marker CD73 and fibroblast marker αSMA. Majority of migration occurred within the first 4days of storage. Our data suggest that endothelial cells on Quarter-DMEK grafts re-distribute during organ-culture storage to re-populate preparation-induced empty bands and after re-distribution, cells may show further migration to the stromal DM side during storage.

Three-Dimensional Characterization of the Normal Human Parafoveal Microvasculature Using Structural Criteria and High-Resolution Confocal Microscopy.

PurposeTo use structural criteria to reconcile the three-dimensional organization and connectivity of the parafoveal microvasculature.MethodsThe parafoveal microvasculature was perfused and labeled in 16 normal human donor eyes for lectin, alpha smooth muscle actin, and filamentous actin. Established structural criteria gathered using confocal microscopy, including vessel diameter, endothelial cell morphology, and presence/density of smooth muscle cells, were used to differentiate arteries, arterioles, capillaries, venules, and veins. Three-dimensional visualization strategies were used to define the connections between retinal arteries and veins within the superficial vascular plexus (SVP), intermediate capillary plexus (ICP), and deep capillary plexus (DCP).ResultsThe parafoveal microvasculature has two different inflow patterns and seven different outflow patterns. The SVP and ICP were connected to retinal arteries by arterioles. Inflow into the DCP occurred only via small arterioles (a1; mean diameter, 8.3 µm) that originated from the ICP. Direct connections between the DCP and retinal arteries were not identified. Each capillary plexus formed its own venule that drained independently or in conjunction with venules from other plexuses into a retinal vein at the level of the ganglion cell layer. For the DCP, a1 was significantly smaller than its draining venule (mean diameter, 18.8 µm; P < 0.001).ConclusionsThe SVP and ICP of the parafoveal microvasculature have both in series and in parallel arterial and venous connections. Arterial supply to the DCP originates from the ICP, but with direct drainage to the retinal vein. These findings may help to develop an understanding of the pattern of retinal lesions characterizing a myriad of retinal vascular diseases.

CoenzymeQ10-Induced Activation of AMPK-YAP-OPA1 Pathway Alleviates Atherosclerosis by Improving Mitochondrial Function, Inhibiting Oxidative Stress and Promoting Energy Metabolism.

Atherosclerosis (AS) is an excessive chronic inflammatory hyperplasia caused by the damage of vascular endothelial cell morphology and function. Changes in mitochondrial internal conformation and increase of reactive oxygen species (ROS) can lead to energy metabolism disorders in mitochondria, which further affects the occurrence of atherosclerosis by impairing vascular endothelial function. Coenzyme Q10 (CoQ10) is one of the components of mitochondrial respiratory chain, which has the functions of electron transfer, reducing oxidative stress damage, improving mitochondrial function and promoting energy metabolism. The main purpose of this study is to investigate the protective effects of CoQ10 against AS by improving mitochondrial energy metabolism. Both in high fat diet (HFD) fed APOE−/− mice and in ox-LDL-treated HAECs, CoQ10 significantly decreased the levels of TG, TC and LDL-C and increased the levels of HDL-C, thus playing a role in regulating lipid homeostasis. Meanwhile, CoQ10 decreased the levels of LDH and MDA and increased the levels of SOD and GSH, thus playing a role in regulating oxidation level. CoQ10 also inhibited the over-release of ROS and increased ATP content to improve mitochondrial function. CoQ10 also decreased the levels of related inflammatory factors (ICAM-1, VCAM-1, IL-6, TNF-α and NLRP3). In order to study the mechanism of the experiment, AMPK and YAP were silenced in vitro. The further study suggested AMPK small interfering RNA (siRNA) and YAP small interfering RNA (siRNA) affected the expression of OPA1, a crucial protein regulating the balance of mitochondrial fusion and division and decreased the therapeutic effects of CoQ10. These results indicated that CoQ10 improved mitochondrial function, inhibited ROS production, promoted energy metabolism and attenuated AS by activating AMPK-YAP-OPA1 pathway. This study provides a possible new mechanism for CoQ10 in the treatment of AS and may bring a new hope for the prevention and treatment of AS in the future.

Heme Synthesis Inhibition Blocks Angiogenesis via Mitochondrial Dysfunction.

SummaryThe relationship between heme metabolism and angiogenesis is poorly understood. The final synthesis of heme occurs in mitochondria, where ferrochelatase (FECH) inserts Fe2+ into protoporphyrin IX to produce proto-heme IX. We previously showed that FECH inhibition is antiangiogenic in human retinal microvascular endothelial cells (HRECs) and in animal models of ocular neovascularization. In the present study, we sought to understand the mechanism of how FECH and thus heme is involved in endothelial cell function. Mitochondria in endothelial cells had several defects in function after heme inhibition. FECH loss changed the shape and mass of mitochondria and led to significant oxidative stress. Oxidative phosphorylation and mitochondrial Complex IV were decreased in HRECs and in murine retina ex vivo after heme depletion. Supplementation with heme partially rescued phenotypes of FECH blockade. These findings provide an unexpected link between mitochondrial heme metabolism and angiogenesis.

The morphology of corneal endothelial cells in long term soft contact lens wearers in Kuala Lumpur

PurposeTo analyse and compare the alterations in corneal endothelium morphology induced by different materials and durations of wearing soft contact lenses (CL) among young adults living in Kuala Lumpur. MethodsHealthy soft CL wearers were invited to participate in this study. Visual acuity (VA) was measured using the Snellen chart, and subjective refraction was performed using cross-cylinder technique. Standard ocular assessments were conducted using a slit lamp biomicroscope and morphology of corneal endothelial cells (endothelial cell density, ECD, coefficient variation, COV, hexagonality, HEX and central corneal thickness, CCT) were evaluated using a non-contact specular microscope. Statistical analysis was conducted using ANOVA and data from the right eye only is included. ResultsA total of 72 subjects (32 SiHy and 40 HCL wearers) and 24 non-CL wearers (control) participated in this study. The gender distribution for study subjects was 13 males and 59 females, with a mean age 22.15 ± 1.84 years old. The mean refraction was −1.86 ± 1.25DS. The duration of wearing CL ranged from 1 to 9 years. Subjects were later divided into 2 groups following duration of CL wear: Group 1 (<5 years) and Group 2 (≥5 years) for analysis purposes. Statistical analysis showed significant alterations in ECD, COV and HEX of CL wearers (p < 0.05), with more changes found in HCL and Group 2 wearers. No significant change was found in CCT. ConclusionThis study concludes that soft CL wear induced alterations in the morphology of corneal endothelial cells. Contact lens material and duration of CL wear (in years) are factors that affect the alterations. Optometrists are recommended to regularly evaluate the morphology of corneal endothelial cells in CL wearers and provide necessary intervention when required.

Evaluation of Corneal Structure and Endothelial Morphological Characteristics in Type 2 Diabetic and Non-Diabetic Patients.

PurposeTo compare corneal structure and endothelial morphological features between patients with type 2 diabetes mellitus (DM) and non-diabetic patients; and determine if the DM duration, glycated hemoglobin (HbA1c) levels, and diabetic retinopathy (DR) stage affect corneal morphological properties.Patients and MethodsSixty diabetic patients and 47 age- and sex-matched controls were enrolled in this cross-sectional study. DM group was analyzed according disease duration, HbA1c levels, and presence of retinopathy. Endothelial cell density (ECD) and morphology (average and coefficient of variation [CV] of cell size, percentage of hexagonal cells) were recorded using non-contact specular microscopy. Central corneal thickness (CCT) and corneal volume were measured by scheimpflug tomography. Univariate and multivariate linear regression analyses were performed to evaluate the relationship between demographical, clinical, and ocular variables with CCT and ECD.ResultsThere were no statistically significant differences in the endothelium cell density or morphology between DM and non-DM groups (p>0.05). Also, there was no statistical difference between groups for CCT or corneal volume (p>0.05). Multivariate linear regression analysis showed that older age (p=0.028) was significantly associated with lower ECD; CCT was found to be significantly greater in males (p<0.001) and positively associated with corneal volume (p<0.001).ConclusionThe present study did not find any statistically significant differences between the corneal structural and endothelial characteristics of diabetic and non-diabetic subjects; other demographical parameters, such as age and gender, seem to be more determinant for the corneal properties.

Evaluation of reconstructed human corneal endothelium sheets made with porcine Descemet's membrane in vitro and in vivo

PurposeTo identify the feasibility of reconstructing corneal endothelial sheets by seeding non-infected monoclonal human corneal endothelial cells (HCECs) onto porcine Descemet's membrane (DM) and verifying the function in vitro and in vivo. MethodsDenuded porcine DM was decellularized for haematoxylin and eosin staining, and DNA was removed via incubation with ethylene glycol diglycidyl ether (EGDE). The physical properties of the incubated DMs were evaluated and compared to those of unincubated DMs. The non-infected monoclonal HCECs were examined by chromosome analysis and the cell proliferation was evaluated by BrdU-labelling. Then HCECs at passage 30 were then seeded on the DM and cultured for approximately 5 days. The cell growth, density and expression of the sodium-potassium adenosine triphosphatase (Na+/K+-ATPase), the tight-junction-associated protein zonula occludens (ZO-1) and acetylated alpha tubulin were examined by electron microscopy and immunocytochemistry to compare HCECs cultivated on porcine DM and those cultured in vitro. Cells on the reconstructed HCEC sheets were labeled with DiI, and the sheets were subsequently transplanted into cat eyes via DM endothelial keratoplasty (DMEK). The corneal transparency, thickness, anterior segment, and HCEC density were monitored in vivo, and the corneal endothelial cell morphology and histological structure were examined ex vivo 98 days after surgery. ResultsNo significant differences were observed in the elongation at break of the DMs and the thickness of the DMs incubated with EGDE compared to those of the unincubated DMs (P > 0.05). Results of chromosome analysis shown the number of the HCEC cell line was still 46 and no abnormal chromosome structure was found. BrdU-labelling shown the HCECs stopped proliferating after 5 days and the cells formed a single layer. The cells transferred to porcine DM formed tight connections with the substrate and generated layers of hexagonal cells on day 5. Adjacent cells cultivated on DM were closely attached to each other, tightly adhered to the porcine DM and expressed the Na+/K+-ATPase, ZO-1 protein and acetylated alpha tubulin, as did HCECs cultured in vitro. In addition, the HCEC density on DMs was 3020.14 ± 52.30 cells/mm2. After surgery, the corneas gradually became transparent, and the thickness decreased to 525.33 ± 56.23 μm at day 98 after the transplantation, while the control corneas showed consistent oedema during the monitoring period. The HCEC density was 2521.60 ± 78.24 cells/mm2 in vivo 98 days after transplantation. The histological results showed that the DiI-labeled cells were dense in the transplanted area and had a hexagonal or polygonal morphology and a normal ultrastructure; adjacent cells were closely attached to each other and tightly adhered to the porcine DM. ConclusionsSeeding non-infected monoclonal HCECs on porcine DM could reconstruct functional corneal endothelial sheets. These results may help uncover new applications for tissue-engineered endothelium in endothelial keratoplasty.

Designer Descemet Membranes Containing PDLLA and Functionalized Gelatins as Corneal Endothelial Scaffold.

Corneal blindness is the fourth leading cause of visual impairment. Of specific interest is blindness due to a dysfunctional corneal endothelium which can only be treated by transplanting healthy tissue from a deceased donor. Unfortunately, corneal supply does not meet the demand with only one donor for every 70 patients. Therefore, there is a huge interest in tissue engineering of grafts consisting of an ultra-thin scaffold seeded with cultured endothelial cells. The present research describes the fabrication of such artificial Descemet membranes based on the combination of a biodegradable amorphous polyester (poly (d,l-lactic acid)) and crosslinkable gelatins. Four different crosslinkable gelatin derivatives are compared in terms of processing, membrane quality, and function, as well as biological performance in the presence of corneal endothelial cells. The membranes are fabricated through multi-step spincoating, including a sacrificial layer to allow for straightforward membrane detachment after production. As a consequence, ultrathin (<1µm), highly transparent (>90%), semi-permeable membranes could be obtained with high biological potential. The membranes supported the characteristic morphology and correct phenotype of corneal endothelial cells while exhibiting similar proliferation rates as the positive control. As a consequence, the proposed membranes prove to be a promising synthetic alternative to donor tissue.

Density and morphology of corneal endothelial cell after phacoemulsification using ringer lactate versus balanced salt solution as irrigating solutions

AIM: To compare the difference in corneal endothelial cell density and morphology after phacoemulsification using ringer lactate (RL) and balanced salt solution (BSS) irrigating solutions. METHODS: The prospective randomized controlled trial study was conducted between February 2017 and April 2017 in Dr. YAP Eye Hospital, Yogyakarta, Indonesia. There were a total of 52 subjects (52 eyes) who were senile cataract patients further grouped into two, 26 patients undergoing the phacoemulsification procedure using RL irrigating solution and the other 26 patients with BSS irrigating solution, both conducted by one operator. On the 1, 7, and 28d post operative, an evaluation was done to measure the density and corneal endothelial cell morphology, as well as the variable of inflammation in the two groups. RESULTS: Fifty-two eyes had undergone phacoemulsification with posterior intraocular lens implantation. Both groups were evaluated for the endothelial cell reduction and corneal endothelial cell morphology change, along with postoperative inflammation. On the 28d postoperative, endothelial cell reduction in the BSS group (173.96 cell/mm2, 8.12%) was lower than the RL group (253.20 cell/mm2, 10.25%), percentage of corneal endothelial cell variation coefficient increase in the BSS group (2.92%, 8.36%) was lower compared to the RL group (3.42%, 9.96%), decrease of hexagonal cells of corneal endothelium cells presentation percentage in the BSS group (4.30%, 8.17%) was lower compared to the RL group (4.84%, 8.97%), and the percentage increase of central corneal thickness in the BSS group (4.69 μm, 0.89%) was almost equal to the RL group (4.53 μm, 0.90%). All of the results regarding difference in density and corneal cell endothelium morphology between the two groups did not reveal any statistically significant difference (P&gt;0.05). Inflammatory variable in the two groups were even. CONCLUSION: BSS and RL were equal in their capability of maintaining endothelial cell loss and endothelial cell morphologic change in senile cataract patients after phacoemulsification.

Effects of mitomycin C at different exposure time on the morphology of corneal endothelial cells in LASEK surgery

AIM: To investigate the effects of mitomycin C (MMC) on corneal endothelial cells at different exposure time during laser-assisted subepithelial keratomileusis (LASEK). METHODS: Patients who received LASEK were included and divided into Group T1 (15s≤t≤45s) and T2 (5s&lt;t≤70s) based on the exposure time of MMC with corneal stroma. Cell density (CD) of corneal endothelial cells in central and peripheral cornea, average cell area (ACA), area standard deviation (SD), coefficient of variation (CV) and percentage of hexagonal cells (HEX) before surgery and at 1wk, 1, 3 and 6mo after surgery were compared between and within the groups. RESULTS: A total of 196 eyes of 98 patients were included with 98 eyes in Group T1 and 98 eyes in Group T2. With the exception of peripheral ACA, central CV and HEX in Group T1 and T2, which significantly changed at 1wk after the surgery (P&lt;0.05), there was no significant difference in central and peripheral parameters within both groups or between the groups (P&gt;0.05). CONCLUSION: Although transient acute changes in central CV and HEX and peripheral ACA were observed, there is no significant effect on the long-term corneal health status when MMC is applied in a reasonable time range in LASEK surgery.

Corneal Stromal Filler Injection as a Novel Approach to Correct Presbyopia-An Ex Vivo Pilot Study.

PurposeTo evaluate the ex vivo feasibility of corneal stromal filler injection to create bifocality to correct presbyopia by flattening the central posterior corneal surface and thus increase refractive power.MethodsFemtosecond laser-assisted corneal stromal pockets of varying diameters close to the posterior corneal curvature were cut into rabbit eyes ex vivo. Subsequently, hyaluronic acid was injected to flatten the central posterior curvature. Refractive parameters were determined using perioperatively acquired three-dimensional optical coherence tomography (OCT) scans. Using micrometer-resolution OCT, corneal endothelial cell morphology and density were evaluated.ResultsFollowing filler injection into the corneal stromal pockets, a fair volume-dependent increase of central refractive power up to 4 diopters (dpt) was observed. Unremarkable refractive changes of the peripheral posterior (3 mm, 0.20 ± 0.11 dpt; 2 mm, 0.11 ± 0.10 dpt) and the anterior corneal curvature (3 mm, 0.20 ± 0.34 dpt; 2 mm, 0.33 ± 0.31 dpt) occurred. Only negligible changes in astigmatism were observed. Different sizes of optical zones could be established. Furthermore, no alterations of corneal endothelial morphology or endothelial cell density (2831 ± 356 cells/mm2 vs. 2734 ± 292 cells/mm2; P = 0.552) due to the adjacent laser treatment were observed.ConclusionsThe ex vivo investigations proved the principle of injecting a filler material into femtosecond laser-created corneal stromal pockets close to the posterior corneal curvature as an efficacious, individually adjustable, and novel approach to correct presbyopia without ablating corneal tissue.Translational RelevanceDue to the aging population worldwide, presbyopia is an increasing problem; thus, our study may encourage further exploration to extend the treatment spectrum of clinically used femtosecond laser systems to correct presbyopia.

Extracellular vesicles in diabetes mellitus induce alterations in endothelial cell morphology and migration

BackgroundInflammation-related atherosclerotic peripheral vascular disease is a major end organ complication of diabetes mellitus that results in devastating morbidity and mortality. Extracellular vesicles (EVs) are nano-sized particles that contain molecular cargo and circulate in the blood. Here, we examined EV protein cargo from diabetic individuals and whether these EVs cause functional changes in endothelial cells.MethodsWe quantified inflammatory protein levels in plasma-derived EVs from a longitudinal cohort of euglycemic and diabetic individuals and used in vitro endothelial cell biological assays to assess the functional effects of these EVs with samples from a cross-sectional cohort.ResultsWe found several significant associations between EV inflammatory protein levels and diabetes status. The angiogenic factor, vascular endothelial growth factor A (VEGF-A), was associated with diabetes status in our longitudinal cohort. Those with diabetes mellitus had higher EV VEGF-A levels compared to euglycemic individuals. Additionally, EV levels of VEGF-A were significantly associated with homeostatic model assessment of insulin resistance (HOMA-IR) and β-cell function (HOMA-B). To test whether EVs with different inflammatory cargo can demonstrate different effects on endothelial cells, we performed cell migration and immunofluorescence assays. We observed that EVs from diabetic individuals increased cell lamellipodia formation and migration when compared to EVs from euglycemic individuals.ConclusionsHigher levels of inflammatory proteins were found in EVs from diabetic individuals. Our data implicate EVs as playing important roles in peripheral vascular disease that occur in individuals with diabetes mellitus and suggest that EVs may serve as an informative diagnostic tool for the disease.

Fibronectin rescues aberrant phenotype of endothelial cells lacking either CCM1, CCM2 or CCM3.

Loss-of-function variants in CCM1/KRIT1, CCM2, and CCM3/PDCD10 are associated with autosomal dominant cerebral cavernous malformations (CCMs). CRISPR/Cas9-mediated CCM3 inactivation in human endothelial cells (ECs) has been shown to induce profound defects in cell-cell interaction as well as actin cytoskeleton organization. We here show that CCM3 inactivation impairs fibronectin expression and consequently leads to reduced fibers in the extracellular matrix. Despite the complexity and high molecular weight of fibronectin fibrils, our in vitro model allowed us to reveal that fibronectin supplementation restored aberrant spheroid formation as well as altered EC morphology, and suppressed actin stress fiber formation. Yet, fibronectin replacement neither enhanced the stability of tube-like structures nor inhibited the survival advantage of CCM3-/- ECs. Importantly, CRISPR/Cas9-mediated introduction of biallelic loss-of-function variants into either CCM1 or CCM2 demonstrated that the impaired production of a functional fibronectin matrix is a common feature of CCM1-, CCM2-, and CCM3-deficient ECs.

Elasticity of fiber meshes from multiblock copolymers influences endothelial cell behavior.

The behavior of endothelial cells is remarkably influenced by the physical and biochemical signals from their surrounding microenvironments. Here, the elasticity of fiber meshes was studied as a design parameter of substrates for endothelial cells in order to modulate angiogenesis. Human umbilical vein endothelial cells (HUVECs) were cultured on electrospun fiber meshes made from polyetheresterurethane (PEEU), differing in their elasticity. Cell morphology, proliferation, migration and angiogenesis of endothelial cells on the degradable substrate meshes were characterized. The aspect ratio of HUVECs cultured on the fiber meshes from PEEU materials increased with increasing stiffness of the materials. HUVECs cultured on fiber meshes with high stiffness (Young's modulus E = 4.5±0.8 MPa) presented a higher proliferation rate and significantly faster migration velocity, as well as higher tube formation capability than the cells cultured on fiber meshes with low stiffness (E = 2.6±0.8 MPa). These results suggested that tuning the fiber meshes' elasticity might be a potential strategy for modulating the formation or regeneration of blood vessels.