Bionic eye system mimicking microfluidic structure and intraocular pressure for glaucoma surgery training.

Among increasing eye diseases, glaucoma may hurt the optic nerves and lead to vision loss, the treatment of which is to reduce intraocular pressure (IOP). In this research, we introduce a new concept of the surgery simulator for Minimally Invasive Glaucoma Surgery (MIGS). The concept is comprised of an anterior eye model and a fluidic circulatory system. The model made of flexible material includes a channel like the Schlemm's canal (SC) and a membrane like the trabecular meshwork (TM) covering the SC. The system can monitor IOP in the model by a pressure sensor. In one of the MIGS procedures, the TM is cleaved to reduce the IOP. Using the simulator, ophthalmologists can practice the procedure and measure the IOP. First, considering the characteristics of human eyes, we defined requirements and target performances for the simulator. Next, we designed and manufactured the prototype. Using the prototype, we measured the IOP change before and after cleaving the TM. Finally, we demonstrated the availability by comparing experimental results and target performances. This simulator is also expected to be used for evaluations and developments of new MIGS instruments and ophthalmic surgery robots in addition to the surgical training of ophthalmologists.

Identification of Estrogen Signaling in a Prioritization Study of Intraocular Pressure-Associated Genes.

Elevated intraocular pressure (IOP) is the only modifiable risk factor for primary open-angle glaucoma (POAG). Herein we sought to prioritize a set of previously identified IOP-associated genes using novel and previously published datasets. We identified several genes for future study, including several involved in cytoskeletal/extracellular matrix reorganization, cell adhesion, angiogenesis, and TGF-ß signaling. Our differential correlation analysis of IOP-associated genes identified 295 pairs of 201 genes with differential correlation. Pathway analysis identified ß-estradiol as the top upstream regulator of these genes with ESR1 mediating 25 interactions. Several genes (i.e., EFEMP1, FOXC1, and SPTBN1) regulated by ß-estradiol/ESR1 were highly expressed in non-glaucomatous human trabecular meshwork (TM) or Schlemm's canal (SC) cells and specifically expressed in TM/SC cell clusters defined by single-cell RNA-sequencing. We confirmed ESR1 gene and protein expression in human TM cells and TM/SC tissue with quantitative real-time PCR and immunofluorescence, respectively. 17ß-estradiol was identified in bovine, porcine, and human aqueous humor (AH) using ELISA. In conclusion, we have identified estrogen receptor signaling as a key modulator of several IOP-associated genes. The expression of ESR1 and these IOP-associated genes in TM/SC tissue and the presence of 17ß-estradiol in AH supports a role for estrogen signaling in IOP regulation.

Altered Expression of Extracellular Vesicles miRNAs from Primary Human Trabecular Meshwork Cells Induced by Transforming Growth Factor-ß2.

Primary open-angle glaucoma (POAG) is tightly related with extracellular matrix (ECM) remodeling of human trabecular meshwork cells (HTMCs). Transforming growth factor-ß2 (TGF-ß2) can induce ECM remodeling. The aim of the study was to investigate the microRNAs (miRNAs) expression changes of extracellular vesicles (EVs) derived from HTMCs treated with TGF-ß2. EVs were isolated from HTMCs supernatant cultured for 24 h with TGF-ß2. The morphology of EVs pellets was examined by transmission electron microscopy. Nanoparticle tracking analysis used to demonstrate the particle size distribution. Total EVs RNAs were extracted for subsequent miRNA gene chip analysis to investigate differentially expressed miRNAs between the controls and treatment cells. Gene Ontology (GO) annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were used to predict potential target and validate possible functions of the miRNAs. There were 23 miRNAs upregulated and 3 miRNAs downregulated and 469,102, and 94 GO terms involved in biological processes, cellular components, and molecular function for the possible functions of the 26 miRNAs. These findings indicate that TGF-ß2 may alter EVs miRNAs expression to participate in the pathogenesis of POAG. They may provide significant information for potential biomarkers for POAG diagnosis and treatment.

Influence of Anti-Glaucoma Drugs on Uptake of Extracellular Vesicles by Trabecular Meshwork Cells.

BACKGROUND: Extracellular vesicles (EVs) are capable of manipulating cellular functions for the maintenance of biological homeostasis and disease progression, such as in glaucoma disease. These nano-particles carry a net negative surface charge under physiological conditions that can contribute to EVs:EVs interaction and their uptake by target cells. PURPOSE: To investigate the effect of glaucoma drugs on EVs physicochemical characters and the implications for their uptake by trabecular meshwork (TM) cells. METHODS: TM or non-pigmented ciliary epithelium (NPCE) cells derived EVs were incubated with commercial anti-glaucoma formulation, Timolol maleate, Brinzolamide or Benzalkonium Cl and their size and zeta potential (ZP) and physical interactions of EVs derived from NPCE cells and TM cells were evaluated. The contribution of EVs interactions to up-take by TM cells was examined using fluorescence-activated cell sorting. RESULTS: EVs size and ZP were affected by the ionic strength of the buffer rather than EVs type. Commercial glaucoma eye drops, including ß-blocker, α-2-agonist and prostaglandin analogs, reduced NPCE EVs ZP, whereas exposure of EVs to carbonic anhydrase inhibitor caused an increase in the ZP. A correlation was found between increased ZP values and increased NPCE EVs uptake by TM cells. We were able to show that Benzalkonium chloride stands behind this ZP effect and not Timolol or Brinzolamide. CONCLUSION: Altogether, our findings demonstrate that EVs size, surface membrane charge, and ionic strength of the surrounding have an impact on EVs:EVs interactions, which affect the uptake of NPCE EVs by TM cells.

[The trabecular meshwork: Structure, function and clinical implications. A review of the littérature (French translation of the article)]. / Le trabéculum : structure, fonction et implications cliniques. Une revue de la littérature.

Glaucoma is a blinding optic neuropathy, the main risk factor for which is increased intraocular pressure (IOP). The trabecular meshwork, located within the iridocorneal angle, is the main pathway for drainage of aqueous humor (AH) out of the eye, and its dysfunction is responsible for the IOP elevation. The trabecular meshwork is a complex, fenestrated, three-dimensional structure composed of trabecular meshwork cells (TMC) interdigitated into a multilayered organization within the extracellular matrix (ECM). The purpose of this literature review is to provide an overview of current understanding of the trabecular meshwork and its pathophysiology in glaucoma. Thus, we will present the main anatomical and cellular bases for the regulation of aqueous humor outflow resistance, the pathophysiological mechanisms involved in trabecular dysfunction in the various types of glaucoma, as well as current and future therapeutic strategies targeting the trabecular meshwork.

The trabecular meshwork: Structure, function and clinical implications. A review of the literature.

Glaucoma is a blinding optic neuropathy, the main risk factor for which is increased intraocular pressure (IOP). The trabecular meshwork, located within the iridocorneal angle, is the main pathway for drainage of aqueous humor (AH) out of the eye, and its dysfunction is responsible for the IOP elevation. The trabecular meshwork is a complex, fenestrated, three-dimensional structure composed of trabecular meshwork cells (TMC) interdigitated into a multilayered organization within the extracellular matrix (ECM). The purpose of this literature review is to provide an overview of current understanding of the trabecular meshwork and its pathophysiology in glaucoma. Thus, we will present the main anatomical and cellular bases for the regulation of aqueous humor outflow resistance, the pathophysiological mechanisms involved in trabecular dysfunction in the various types of glaucoma, as well as current and future therapeutic strategies targeting the trabecular meshwork.

Adipose-derived stem cells integrate into trabecular meshwork with glaucoma treatment potential.

The trabecular meshwork (TM) is an ocular tissue that maintains intraocular pressure (IOP) within a physiologic range. Glaucoma patients have reduced TM cellularity and, frequently, elevated IOP. To establish a stem cell-based approach to restoring TM function and normalizing IOP, human adipose-derived stem cells (ADSCs) were induced to differentiate to TM cells in vitro. These ADSC-TM cells displayed a TM cell-like genotypic profile, became phagocytic, and responded to dexamethasone stimulation, characteristic of TM cells. After transplantation into naive mouse eyes, ADSCs and ADSC-TM cells integrated into the TM tissue, expressed TM cell markers, and maintained normal IOP, outflow facility, and extracellular matrix. Cell migration and affinity results indicated that the chemokine pair CXCR4/SDF1 may play an important role in ADSC-TM cell homing. Our study demonstrates the possibility of applying autologous or allogeneic ADSCs and ADSC-TM cells as a potential treatment to restore TM structure and function in glaucoma.

An injectable peptide hydrogel for reconstruction of the human trabecular meshwork.

Glaucoma is a leading cause of irreversible blindness worldwide. Current treatments of glaucoma involve lowering the IOP by means of decreasing aqueous humor production or increasing non-trabecular aqueous humor outflow with the help of IOP-lowering eye drops, nanotechnology enabled glaucoma drainage implants, and trabeculectomy. However, there is currently no effective and permanent cure for this disease. In order to investigate new therapeutic strategies, three dimensional (3D) biomimetic trabecular meshwork (TM) models are in demand. Therefore, we adapted MAX8B, a peptide hydrogel system to bioengineer a 3D trabecular meshwork scaffold. We assessed mechanical and bio-instructive properties of this engineered tissue matrix by using rheological analysis, 3D cell culture and imaging techniques. The scaffold material exhibited shear-thinning ability and biocompatibility for proper hTM growth and proliferation indicating a potential utilization as an injectable implant. Additionally, by using a perfusion system, MAX8B scaffold was tested as an in vitro platform for investigating the effect of Dexamethasone (Dex) on trabecular meshwork outflow facility. The physiological response of hTM cells within the scaffold to Dex treatment clearly supported the effectiveness of this 3D model as a drug-testing platform, which can accelerate discovery of new therapeutic targets for glaucoma. STATEMENT OF SIGNIFICANCE: Artificial 3D-TM (3-dimentional Trabecular Meshwork) developed here with hTM (human TM) cells seeded on peptide-hydrogel scaffolds exhibits the mechanical strength and physiological properties mimicking the native TM tissue. Besides serving a novel and effective 3D-TM model, the MAX8B hydrogel could potentially function as an injectable trabecular meshwork implant.

Trabecular Meshwork TREK-1 Channels Function as Polymodal Integrators of Pressure and pH.

Purpose: The concentration of protons in the aqueous humor (AH) of the vertebrate eye is maintained close to blood pH; however, pathologic conditions and surgery may shift it by orders of magnitude. We investigated whether and how changes in extra- and intracellular pH affect the physiology and function of trabecular meshwork (TM) cells that regulate AH outflow. Methods: Electrophysiology, in conjunction with pharmacology, gene knockdown, and optical recording, was used to track the pH dependence of transmembrane currents and mechanotransduction in primary and immortalized human TM cells. Results: Extracellular acidification depolarized the resting membrane potential by inhibiting an outward K+-mediated current, whereas alkalinization hyperpolarized the cells and augmented the outward conductance. Intracellular acidification with sodium bicarbonate hyperpolarized TM cells, whereas removal of intracellular protons with ammonium chloride depolarized the membrane potential. The effects of extra- and intracellular acid and alkaline loading were abolished by quinine, a pan-selective inhibitor of two-pore domain potassium (K2P) channels, and suppressed by shRNA-mediated downregulation of the mechanosensitive K2P channel TREK-1. Extracellular acidosis suppressed, whereas alkalosis facilitated, the amplitude of the pressure-evoked TREK-1-mediated outward current. Conclusions: These results demonstrate that TM mechanotransduction mediated by TREK-1 channels is profoundly sensitive to extra- and intracellular pH shifts. Intracellular acidification might modulate aqueous outflow and IOP by stimulating TREK-1 channels.

Differentiation of microfluidic-encapsulated trabecular meshwork mesenchymal stem cells into insulin producing cells and their impact on diabetic rats.

Tissue and stem cell encapsulation andtransplantation were considered as promising tools in the treatment of patients with diabetes mellitus. The aim of this study was to evaluate the effect of microfluidic encapsulation on the differentiation of trabecular meshwork mesenchymal stem cells (TM-MSC), into insulin-producing cells (IPCs) both in vitro and in vivo. The presence of differentiated cells in microfibers (three dimensional [3D]) and tissue culture plates (TCPS; two dimensional [2D]) culture was evaluated by detecting mRNA and protein expression of pancreatic islet-specific markers as well as measuring insulin release of cells in response to glucose challenges. Finally, semi-differentiated cells in microfibers (3D) and 2D cultures were used to control the glucose level in diabetic rats. The results of this study showed that MSCs differentiated in alginate microfibers (fabricated by microfluidic device) express more Pdx-1 mRNA (1.938-fold, p-value: 0.0425) and Insulin mRNA (2.841-fold, p-value: 0.0001) compared with those cultured on TCPS. Furthermore, cell encapsulation in microfluidic derived microfibers decreased the level of blood glucose in diabetic rats. The approach used in this study showed the possibility of alginate microfibers as a matrix for differentiation of TM-MSCs (as a new source) into IPCs. In addition, it could minimize different steps in stem cell differentiation, handling, and encapsulation, which lead to loss of an unlimited number of cells.

Histopathological trabecular meshwork remodeling after cataract surgery detected with an advanced image analyzer.

PURPOSE: To compare the histopathological morphometry of the trabecular meshwork and ciliary processes in pseudophakic eyes and phakic eyes using advanced image analyzer technology. SETTING: McGill University, Montreal, Quebec, Canada. DESIGN: Retrospective case series. METHODS: Thirty-five pseudophakic eyes and 25 phakic eyes were sectioned and converted into digital slides. The total trabecular meshwork area and the ciliary body stroma were demarcated. The area of the trabecular meshwork, cellular and noncellular trabecular meshwork compartments, trabecular space, distance from scleral spur to inner uveal trabecular portion, and degree of fibrosis of the ciliary processes were evaluated. RESULTS: The trabecular meshwork area was larger in the pseudophakic group than the phakic group (P = .03). Furthermore, a trend of larger trabecular space recorded was seen in the pseudophakic group than the phakic group (P = .14). No differences in the proportion of cellular (P = .88) and noncellular trabecular meshwork compartments (P = .4) were seen between groups. The scleral spur to inner uveal trabecular portion distance was longer in the pseudophakic group than the phakic group (P = .008) and correlate with the trabecular meshwork area (P = .0001, r = 0.56). In the ciliary processes, a higher degree of fibrosis was measured in the pseudophakic group than the phakic group (P = .02). CONCLUSIONS: There were significant histopathological changes in the trabecular meshwork and higher fibrosis in the ciliary processes in pseudophakic eyes compared with phakic eyes. These findings support the hypothesis that trabecular meshwork remodeling after cataract surgery is involved in lowering intraocular pressure.

HDAC Inhibitor-Mediated Epigenetic Regulation of Glaucoma-Associated TGFß2 in the Trabecular Meshwork.

PURPOSE: Elevated intraocular pressure (IOP) in primary open-angle glaucoma (POAG) results from glaucomatous damage to the trabecular meshwork (TM). The glaucoma-associated factor TGFß2 is increased in aqueous humor and TM of POAG patients. We hypothesize that histone acetylation has a role in dysregulated TGFß2 expression. METHODS: Protein acetylation was compared between nonglaucomatous TM (NTM) and glaucomatous TM (GTM) cells using Western immunoblotting (WB). Nonglaucomatous TM cells were treated with 10 nM thailandepsin-A (TDP-A), a potent histone deacetylase inhibitor for 4 days. Total and nuclear proteins, RNA, and nuclear protein-DNA complexes were harvested for WB, quantitative PCR (qPCR), and chromatin immunoprecipitation (ChIP) assays, respectively. Paired bovine eyes were perfused with TDP-A versus DMSO, or TDP-A versus TDP-A plus the TGFß pathway inhibitor LY364947 for 5 to 9 days. Intraocular pressure, TM, and perfusate proteins were compared. RESULTS: We found increased acetylated histone 3 and total protein acetylation in the GTM cells and TDP-A treated NTM cells. Chromatin immunoprecipitation assays showed that TDP-A induced histone hyperacetylation associated with the TGFß2 promoter. This change of acetylation significantly increased TGFß2 mRNA and protein expression in NTM cells. In perfusion-cultured bovine eyes, TDP-A increased TGFß2 in the perfusate as well as elevated IOP. Histologic and immunofluorescent analyses showed increased extracellular matrix and cytoskeletal proteins in the TM of TDP-A treated bovine eyes. Cotreatment with the TGFß pathway inhibitor LY364947 blocked TDP-A-induced ocular hypertension. CONCLUSIONS: Our results suggest that histone acetylation has an important role in increased expression of the glaucoma-associated factor TGFß2. Histone hyperacetylation may be the initiator of glaucomatous damage to the TM.

The autophagic lysosomal system in outflow pathway physiology and pathophysiology.

Malfunction of the trabecular meshwork (TM)/schlemm's canal (SC) conventional outflow pathway is associated with elevated intraocular pressure (IOP) and, therefore, increased risk of developing glaucoma, a potentially blinding disease affecting more than 70 million people worldwide. This TM/SC tissue is subjected to different types of stress, including mechanical, oxidative, and phagocytic stress. Long-term exposure to these stresses is believed to lead to a progressive accumulation of damaged cellular and tissue structures causing permanent alterations in the tissue physiology, and contribute to the pathologic increase in aqueous humor (AH) outflow resistance. Autophagy is emerging as an essential cellular survival mechanism against a variety of stressors. In addition to performing basal functions, autophagy acts as a cellular survival pathway and represents an essential mechanism by which organisms can adapt to acute stress conditions and repair stress-induced damage. A decline in autophagy has been observed in most tissues with aging and has been considered responsible, at least in part, for the accumulation of damaged cellular components in almost all tissues of aging organisms. Dysfunction in the autophagy pathway is associated with several human diseases, from infectious diseases to cancer and neurodegeneration. In this review, we will summarize our current knowledge of the emerging roles of autophagy in outflow tissue physiology and pathophysiology, including novel evidence suggesting compromised autophagy in the glaucomatous outflow pathway.

23-gauge transconjunctival vitrectomy in eyes with pre-existing functioning filtering blebs.

BACKGROUND: We investigated the outcome of 23-gauge transconjunctival pars plana vitrectomy (23G PPV) for the treatment of vitreoretinal disorder in patients with prior trabeculectomy. METHODS: We retrospectively reviewed medical records of 23G PPV in 11 eyes that had functioning filtering blebs after trabeculectomy. The main outcome measures were the visual acuity, intraocular pressure (IOP) and subconjunctival fluid height in bleb by anterior segment optical coherence tomography (OCT) before and after the surgery. RESULTS: Eyes that had 23G PPV showed improvement of visual acuity after the surgery (P =0.003). Mean IOP was 13.82 mmHg before 23G PPV and 15.82 mmHg at 6 months postoperatively, which was statistically insignificant (P = 0.758). The bleb was observed before and after surgery using anterior segment OCT, and the difference in subconjunctival fluid was not statistically significant (P =0.172). CONCLUSIONS: 23G PPV did not adversely affect bleb function in eyes with prior trabeculectomy.

Matricellular proteins in the trabecular meshwork: review and update.

Abstract Primary open-angle glaucoma (POAG) is a leading cause of blindness worldwide, and intraocular pressure (IOP) is an important modifiable risk factor. IOP is a function of aqueous humor production and aqueous humor outflow, and it is thought that prolonged IOP elevation leads to optic nerve damage over time. Within the trabecular meshwork (TM), the eye's primary drainage system for aqueous humor, matricellular proteins generally allow cells to modulate their attachments with and alter the characteristics of their surrounding extracellular matrix (ECM). It is now well established that ECM turnover in the TM affects outflow facility, and matricellular proteins are emerging as significant players in IOP regulation. The formalized study of matricellular proteins in TM has gained increased attention. Secreted protein acidic and rich in cysteine (SPARC), myocilin, connective tissue growth factor (CTGF), and thrombospondin-1 and -2 (TSP-1 and -2) have been localized to the TM, and a growing body of evidence suggests that these matricellular proteins play an important role in IOP regulation and possibly the pathophysiology of POAG. As evidence continues to emerge, these proteins are now seen as potential therapeutic targets. Further study is warranted to assess their utility in treating glaucoma in humans.

Novel characterization and live imaging of Schlemm's canal expressing Prox-1.

Schlemm's canal is an important structure of the conventional aqueous humor outflow pathway and is critically involved in regulating the intraocular pressure. In this study, we report a novel finding that prospero homeobox protein 1 (Prox-1), the master control gene for lymphatic development, is expressed in Schlemm's canal. Moreover, we provide a novel in vivo method of visualizing Schlemm's canal using a transgenic mouse model of Prox-1-green fluorescent protein (GFP). The anatomical location of Prox-1⁺ Schlemm's canal was further confirmed by in vivo gonioscopic examination and ex vivo immunohistochemical analysis. Additionally, we show that the Schlemm's canal is distinguishable from typical lymphatic vessels by lack of lymphatic vessel endothelial hyaluronan receptor (LYVE-1) expression and absence of apparent sprouting reaction when inflammatory lymphangiogenesis occurred in the cornea. Taken together, our findings offer new insights into Schlemm's canal and provide a new experimental model for live imaging of this critical structure to help further our understanding of the aqueous humor outflow. This may lead to new avenues toward the development of novel therapeutic intervention for relevant diseases, most notably glaucoma.

AMP-activated protein kinase regulates intraocular pressure, extracellular matrix, and cytoskeleton in trabecular meshwork.

PURPOSE: In this study, we investigate how adenosine monophosphate-activated protein kinase (AMPK) affects extracellular matrix (ECM) and cellular tone in the trabecular meshwork (TM), and examine how deletion of its catalytic α2 subunit affects IOP and aqueous humor clearance in mice. METHODS: Human TM tissue was examined for expression of AMPKα1 and AMPKα2, genomically distinct isoforms of the AMPK catalytic subunit. Primary cultured human TM cells were treated for 24 hours with the AMPK activator 5-amino-1-ß-Dffff-ribofuranosyl-imidazole-4-carboxamide (AICAR), under basal or TGF-ß2 stimulatory conditions. Conditioned media (CM) was probed for secreted protein acidic and rich in cysteine (SPARC), thrombospondin-1 (TSP-1), and ECM proteins, and cells were stained for F-actin. Cells underwent adenoviral infection with a dominant negative AMPKα subunit (ad.DN.AMPKα) and were similarly analyzed. Intraocular pressure, central corneal thickness (CCT), and aqueous clearance were measured in AMPKα2-null and wild-type (WT) mice. RESULTS: Both AMPKα1 and AMPKα2 are expressed in TM. AICAR activated AMPKα and suppressed the expression of various ECM proteins under basal and TGF-ß2 stimulatory conditions. AICAR decreased F-actin staining and increased the phospho-total RhoA ratio (Ser188). Transforming growth factor-ß2 transiently dephosphorylated AMPKα. Infection with ad.DN.AMPKα upregulated various ECM proteins, decreased the phospho-total RhoA ratio, and increased F-actin staining. AMPKα2-null mice exhibited 6% higher IOP and decreased aqueous clearance compared with WT mice, without significant differences in CCT or angle morphology. CONCLUSIONS: Collectively, our data identify AMPK as a critical regulator of ECM homeostasis and cytoskeletal arrangement in the TM. Mice that are AMPKα2-null exhibit higher IOPs and decreased aqueous clearance than their WT counterparts.

The effects of tenascin C knockdown on trabecular meshwork outflow resistance.

PURPOSE: Tenascin C (TNC) is a matricellular glycoprotein whose expression in adult tissue is indicative of tissue remodeling. The purpose of the current study was to determine the localization of TNC in trabecular meshwork (TM) tissue and to analyze the effects of TNC on intraocular pressure (IOP). METHODS: Human TM frontal sections were immunostained with anti-TNC and imaged by confocal microscopy. TNC mRNA and protein levels were quantitated in anterior segments perfused at physiological and elevated pressure. Short, hairpin RNA (shRNA) silencing lentivirus targeting full-length TNC (shTNC) was applied to anterior segment perfusion organ cultures. The IOPs and central corneal thickness (CCT) of wild-type, TNC(-/-), and tenascin X (TNX(-/-)) knockout mice were measured. RESULTS: TNC was distributed in the juxtacanalicular (JCT) region of adult human TM, predominantly in the basement membrane underlying the inner wall of Schlemm's canal. Application of shTNC lentivirus to human and porcine anterior segments in perfusion culture did not significantly affect outflow rate. Although TNC was upregulated in response to pressure, there was no difference in outflow rate when shTNC-silenced anterior segments were subjected to elevated pressure. Furthermore, IOPs and CCTs were not significantly different between TNC(-/-) or TNX(-/-) and wild-type mice. CONCLUSIONS: TNC does not appear to contribute directly to outflow resistance. However, TNC immunolocalization in the JCT of adult human eyes suggests that certain areas of the TM are being continuously remodeled with or without an IOP increase.

Endocardial endothelium is a key determinant of force-frequency relationship in rat ventricular myocardium.

We tested the hypothesis that removing endocardial endothelium (EE) negatively impacts the force-frequency relationship (FFR) of ventricular myocardium and dissected the signaling that underlies this phenomenon. EE of rat trabeculae was selectively damaged by brief (<1 s) exposure to 0.1% Triton X-100. Force, intracellular Ca(2+) transient (iCa(2+)), and activity of protein kinase A (PKA) and protein kinase C (PKC) were determined. In control muscles, force and iCa(2+) increased as the stimulation frequency increased in steps of 0.5 Hz up to 3.0 Hz. However, EE-denuded (EED) muscles exhibited a markedly blunted FFR. Neither isoproterenol (ISO; 0.1-5 nmol/l) nor endothelin-1 (ET-1; 10-100 nmol/l) alone restored the slope of FFR in EED muscles. Intriguingly, however, a positive FFR was restored in EED preparations by combining low concentrations of ISO (0.1 nmol/l) and ET-1 (20 nmol/l). In intact muscles, PKA and PKC activity increased proportionally with the increase in frequency. This effect was completely lost in EED muscles. Again, combining ISO and ET-1 fully restored the frequency-dependent rise in PKA and PKC activity in EED muscles. In conclusion, selective damage of EE leads to significantly blunted FFR. A combination of low concentrations of ISO and ET-1 successfully restores FFR in EED muscles. The interdependence of ISO and ET-1 in this process indicates cross-talk between the ß1-PKA and ET-1-PKC pathways for a normal (positive) FFR. The results also imply that dysfunction of EE and/or EE-myocyte coupling may contribute to flat (or even negative) FFR in heart failure.

Transplantation of mesenchymal stem cells promotes tissue regeneration in a glaucoma model through laser-induced paracrine factor secretion and progenitor cell recruitment.

Among bone marrow cells, hematopoietic and mesenchymal components can contribute to repair damaged organs. Such cells are usually used in acute diseases but few options are available for the treatment of chronic disorders. In this study, we have used a laser-induced model of open angle glaucoma (OAG) to evaluate the potential of bone marrow cell populations and the mechanisms involved in tissue repair. In addition, we investigated laser-induced tissue remodeling as a method of targeting effector cells into damaged tissues. We demonstrate that among bone marrow cells, mesenchymal stem cells (MSC) induce trabecular meshwork regeneration. MSC injection into the ocular anterior chamber leads to far more efficient decrease in intraocular pressure (IOP) (p < .001) and healing than hematopoietic cells. This robust effect was attributable to paracrine factors from stressed MSC, as injection of conditioned medium from MSC exposed to low but not to normal oxygen levels resulted in an immediate decrease in IOP. Moreover, MSC and their secreted factors induced reactivation of a progenitor cell pool found in the ciliary body and increased cellular proliferation. Proliferating cells were observed within the chamber angle for at least 1 month. Laser-induced remodeling was able to target MSC to damaged areas with ensuing specific increases in ocular progenitor cells. Thus, our results identify MSC and their secretum as crucial mediators of tissue repair in OAG through reactivation of local neural progenitors. In addition, laser treatment could represent an appealing strategy to promote MSC-mediated progenitor cell recruitment and tissue repair in chronic diseases.