Feasibility demonstration of a device for vitreous liquid biopsy incidental to intravitreal injection.

PURPOSE: VitreoDx is an experimental device enabling push-button collection of a neat vitreous liquid biopsy incidental to an intravitreal injection. We explored the ability of the device to collect a sample usable for proteomic biomarker discovery and testing. DESIGN: Pilot study using ex vivo human eyes. METHODS: Non-vitrectomized, human eyes from nine donors 75-91 years of age were refrigerated in BSS and used within 5 days of death. Four VitreoDx devices fitted with 25G needles, and four staked needle insulin syringes with 30G needles, were inserted at equal intervals through the pars plana of each eye and held in place by a fixture. The sampling mode of each VitreoDx device was triggered to attempt to acquire a liquid biopsy up to 70 µL. The plunger of each insulin syringe was retracted to attempt to obtain a liquid biopsy with a maximum volume of 50 µL. Samples acquired with the VitreoDx were extracted to polypropylene cryovials, refrigerated to -80 ºC, and sent for offsite proteomic analysis by proximity extension assay with a focus on panels containing approved and pipelined drug targets for neovascular disease and inflammatory factors. RESULTS: Of the attempted liquid biopsies with the novel 25G VitreoDx, 92% (66 of 72) resulted in successful acquisition (>25 µL) while 89% (64 of 72) attempted by a traditional 30G needle resulted in a successful acquisition. Sample volume sufficient for proteomics array analysis was acquired by the VitreoDx for every eye. Detectable protein was found for 151 of 166 unique proteins assayed in at least 25% of eyes sampled by VitreoDx. CONCLUSIONS: The high acquisition rate achieved by the prototype was similar to that achieved in previous clinical studies where a standard syringe was used with a 25G needle to biopsy vitreous fluid directly prior to standard intravitreal injection. Successful aspiration rates were likewise high for 30G needles. Together, these suggest that it is possible to routinely acquire liquid vitreous biopsies from patients who typically receive intravitreal injections with an injection device using a standard size needle without a vitreous cutter. Protein analysis shows that proteins of interest survive the sampling mechanism and may have potential to direct care in the future.

Identification of small molecule inhibitors against MMP-14 via High-Throughput screening.

Matrix metalloproteinases (MMPs) are involved in various cellular events in physiology and pathophysiology through endopeptidases activity. The expression levels and activities of most MMPs remain minimal in the normal conditions, whereas some MMPs are significantly activated in pathological conditions such as cancer and neovascularization. Hence, MMPs are considered as both diagnostic markers and potential targets for therapeutic agents. Twenty-three known human MMPs share a similar active site structure with a zinc-binding motif, resulting in lack of specificity. Therefore, the enhancement of target specificity is a primary goal for the development of specific MMP inhibitors. MMP-14 regulates VEGFA/VEGFR2-system through cleavage of the non-functional VEGFR1 in vascular angiogenesis. In this study, we developed a fluorescence-based enzymatic assay using a specific MMP-14 substrate generated from VEGFR1 cleavage site. This well optimized assay was used as a primary screen method to identify MMP-14 specific inhibitors from 1,200 Prestwick FDA-approved drug library. Of ten initial hits, two compounds showed IC50 values below 30 µM, which were further validated by direct binding analysis using surface plasmon resonance (SPR). Clioquinol and chloroxine, both of which contain a quinoline structure, were identified as MMP-14 inhibitors. Five analogs were tested, four of which were found to be completely devoid of inhibitory activity. Clioquinol exhibited selectivity towards MMP-14, as it showed no inhibitory activity towards four other MMPs.

TFOS Lifestyle: Impact of elective medications and procedures on the ocular surface.

The word "elective" refers to medications and procedures undertaken by choice or with a lower grade of prioritization. Patients usually use elective medications or undergo elective procedures to treat pathologic conditions or for cosmetic enhancement, impacting their lifestyle positively and, thus, improving their quality of life. However, those interventions can affect the homeostasis of the tear film and ocular surface. Consequently, they generate signs and symptoms that could impair the patient's quality of life. This report describes the impact of elective topical and systemic medications and procedures on the ocular surface and the underlying mechanisms. Moreover, elective procedures performed for ocular diseases, cosmetic enhancement, and non-ophthalmic interventions, such as radiotherapy and bariatric surgery, are discussed. The report also evaluates significant anatomical and biological consequences of non-urgent interventions to the ocular surface, such as neuropathic and neurotrophic keratopathies. Besides that, it provides an overview of the prophylaxis and management of pathological conditions resulting from the studied interventions and suggests areas for future research. The report also contains a systematic review investigating the quality of life among people who have undergone small incision lenticule extraction (SMILE). Overall, SMILE refractive surgery seems to cause more vision disturbances than LASIK in the first month post-surgery, but less dry eye symptoms in long-term follow up.

Lymphangiogenesis Guidance Mechanisms and Therapeutic Implications in Pathological States of the Cornea.

Corneal lymphangiogenesis is one component of the neovascularization observed in several inflammatory pathologies of the cornea including dry eye disease and corneal graft rejection. Following injury, corneal (lymph)angiogenic privilege is impaired, allowing ingrowth of blood and lymphatic vessels into the previously avascular cornea. While the mechanisms underlying pathological corneal hemangiogenesis have been well described, knowledge of the lymphangiogenesis guidance mechanisms in the cornea is relatively scarce. Various signaling pathways are involved in lymphangiogenesis guidance in general, each influencing one or multiple stages of lymphatic vessel development. Most endogenous factors that guide corneal lymphatic vessel growth or regression act via the vascular endothelial growth factor C signaling pathway, a central regulator of lymphangiogenesis. Several exogenous factors have recently been repurposed and shown to regulate corneal lymphangiogenesis, uncovering unique signaling pathways not previously known to influence lymphatic vessel guidance. A strong understanding of the relevant lymphangiogenesis guidance mechanisms can facilitate the development of targeted anti-lymphangiogenic therapeutics for corneal pathologies. In this review, we examine the current knowledge of lymphatic guidance cues, their regulation of inflammatory states in the cornea, and recently discovered anti-lymphangiogenic therapeutic modalities.

Therapeutic Strategies for Restoring Perturbed Corneal Epithelial Homeostasis in Limbal Stem Cell Deficiency: Current Trends and Future Directions.

Limbal stem cells constitute an important cell population required for regeneration of the corneal epithelium. If insults to limbal stem cells or their niche are sufficiently severe, a disease known as limbal stem cell deficiency occurs. In the absence of functioning limbal stem cells, vision-compromising conjunctivalization of the corneal epithelium occurs, leading to opacification, inflammation, neovascularization, and chronic scarring. Limbal stem cell transplantation is the standard treatment for unilateral cases of limbal stem cell deficiency, but bilateral cases require allogeneic transplantation. Herein we review the current therapeutic utilization of limbal stem cells. We also describe several limbal stem cell markers that impact their phenotype and function and discuss the possibility of modulating limbal stem cells and other sources of stem cells to facilitate the development of novel therapeutic interventions. We finally consider several hurdles for widespread adoption of these proposed methodologies and discuss how they can be overcome to realize vision-restoring interventions.

Artificial intelligence in ophthalmology during COVID-19 and in the post COVID-19 era.

PURPOSE OF REVIEW: To highlight artificial intelligence applications in ophthalmology during the COVID-19 pandemic that can be used to: describe ocular findings and changes correlated with COVID-19; extract information from scholarly articles on SARS-CoV-2 and COVID-19 specific to ophthalmology; and implement efficient patient triage and telemedicine care. RECENT FINDINGS: Ophthalmology has been leading in artificial intelligence and technology applications. With medical imaging analysis, pixel-annotated distinguishable features on COVID-19 patients may help with noninvasive diagnosis and severity outcome predictions. Using natural language processing (NLP) and data integration methods, topic modeling on more than 200 ophthalmology-related articles on COVID-19 can summarize ocular manifestations, viral transmission, treatment strategies, and patient care and practice management. Artificial intelligence for telemedicine applications can address the high demand, prioritize and triage patients, as well as improve at home-monitoring devices and secure data transfers. SUMMARY: COVID-19 is significantly impacting the way we are delivering healthcare. Given the already successful implementation of artificial intelligence applications and telemedicine in ophthalmology, we expect that these systems will be embraced more as tools for research, education, and patient care.

Simultaneous fluorescence imaging of distinct nerve and blood vessel patterns in dual Thy1-YFP and Flt1-DsRed transgenic mice.

Blood vessels and nerve tissues are critical to the development and functionality of many vital organs. However, little is currently known about their interdependency during development and after injury. In this study, dual fluorescence transgenic reporter mice were utilized to observe blood vessels and nervous tissues in organs postnatally. Thy1-YFP and Flt1-DsRed (TYFD) mice were interbred to achieve dual fluorescence in the offspring, with Thy1-YFP yellow fluorescence expressed primarily in nerves, and Flt1-DsRed fluorescence expressed selectively in blood vessels. Using this dual fluorescent mouse strain, we were able to visualize the networks of nervous and vascular tissue simultaneously in various organ systems both in the physiological state and after injury. Using ex vivo high-resolution imaging in this dual fluorescent strain, we characterized the organizational patterns of both nervous and vascular systems in a diverse set of organs and tissues. In the cornea, we also observed the dynamic patterns of nerve and blood vessel networks following epithelial debridement injury. These findings highlight the versatility of this dual fluorescent strain for characterizing the relationship between nerve and blood vessel growth and organization.

Quantification of Angiogenesis and Lymphangiogenesis in the Dual ex vivo Aortic and Thoracic Duct Assay.

BACKGROUND: Lymphatic vessel formation (lymphangiogenesis) plays important roles in cancer metastasis, organ rejection, and lymphedema, but the underlying molecular events remain unclear. Furthermore, despite significant overlap in the molecular families involved in angiogenesis and lymphangiogenesis, little is known about the crosstalk between these processes. The ex vivo aortic ring assay and lymphatic ring assay have enabled detailed studies of vessel sprouting, but harvesting and imaging clear thoracic duct samples remain challenging. Here we present a modified ex vivo dual aortic ring and thoracic duct assay using tissues from dual fluorescence reporter Prox1- GFP/Flt1-DsRed (PGFD) mice, which permit simultaneous visualization of blood and lymphatic endothelial cells. OBJECTIVE: To characterize the concurrent sprouting of intrinsically fluorescent blood and lymphatic vessels from harvested aorta and thoracic duct samples. METHODS: Dual aorta and thoracic duct specimens were harvested from PGFD mice, grown in six types of endothelial cell growth media (one control, five that each lack a specific growth factor), and visualized by confocal fluorescence microscopy. Linear mixed models were used to compare the extent of vessel growth and sprouting over a 28-day period. RESULTS: Angiogenesis occurred prior to lymphangiogenesis in our assay. The control medium generally induced superior growth of both vessel types compared with the different modified media formulations. The greatest decrease in lymphangiogenesis was observed in vascular endothelial growth factor-C (VEGF-C)-devoid medium, suggesting the importance of VEGF-C in lymphangiogenesis. CONCLUSION: The modified ex vivo dual aortic ring and thoracic duct assay represents a powerful tool for studying angiogenesis and lymphangiogenesis in concert.

MMP14-Containing Exosomes Cleave VEGFR1 and Promote VEGFA-Induced Migration and Proliferation of Vascular Endothelial Cells.

Purpose: Investigate the impact matrix metalloproteinase 14 (MMP14) delivered via exosomes produced by corneal fibroblasts on vascular endothelial growth factor receptor 1 (VEGFR1) cleavage on endothelial cells, and other key processes of angiogenesis. Methods: Proteolysis of VEGFR1 and R2 by the catalytic domain of MMP14 was investigated via immunocytochemistry with anti-VEGFR1, anti-VEGFR2, and anti-MMP14 antibodies. Exosomes were isolated via precipitation and serial ultracentrifugation from wild-type (WT) and MMP14 exon4-deficient corneal fibroblasts. Transmission electron microscopy and nanotracking analysis were used to characterize the isolated exosomes. The presence of MMP14 in exosomes from WT fibroblasts was confirmed by Western blotting. VEGFR1 cleavage upon treatment with WT-derived exosomes, Δexon4-derived exosomes, or the pan-MMP inhibitor GM60001 was examined via in vitro proteolysis analysis using recombinant mouse (rm) VEGFR1/R2. Endothelial cell migration and proliferation were investigated using a Boyden chamber assay and BrdU incorporation, respectively. Results: WT-derived exosomes specifically cleaved rmVEGFR1 in vitro, whereas Δexon4-derived exosomes did not. Treatment with the pan-MMP inhibitor GM6001 effectively inhibited VEGFR1 cleavage by WT-derived exosomes, confirming the role of MMP14 in this cleavage. WT-derived exosomes induced greater endothelial cell migration (P < 0.01) and proliferation (P < 0.5) compared to Δexon4-derived exosomes. Conclusions: MMP14-containing exosomes may be involved in the regulation of corneal neovascularization through degradation of VEGFR1 and VEGFA-induced endothelial cell proliferation and migration.

Direct and Indirect Flap Measurements in Femtosecond Laser-Assisted In Situ Keratomileusis.

PURPOSE: To compare direct and indirect LASIK flap thickness measurements using ultrasound and Scheimpflug technology. METHODS: Eighty-two eyes treated with laser-assisted in situ keratomileusis refractive surgery using a femtosecond laser (IntraLase FS150) were prospectively included in the study. Flap thickness was set to 115 µm. Corneal flap thickness was measured using the direct method-ie, ultrasound pachymetry immediately after flap construction in the presence of cavitation bubbles-and indirect methods, with subtraction of intraoperative post-lift corneal thickness measured using ultrasound pachymetry (intrastroma) from preoperative central corneal thickness using ultrasound (Indirect-US) or Scheimpflug thinnest pachymetry (Indirect-Scheimpflug). RESULTS: Mean flap thickness was overestimated using the indirect methods, Indirect-US and Indirect-Scheimpflug (122.6 ± 24.5 µm and 128.1 ± 26.1 µm, respectively; P < 0.0060 and P < 0.0001, respectively). There were no significant correlations between the direct and indirect methods. Indirect-Scheimpflug was significantly higher (P = 0.0122) than Indirect-US. The closest average flap thickness compared with the set parameter of 115 µm was that of the direct method (115.6 ± 8.6 µm; 95% confidence interval: -1.3 to 2.5; P = 0.5163). The direct method provided the lowest SD of all groups (SD: 8.64). CONCLUSIONS: The direct method of flap thickness measurement was the most comparable to the set parameter compared with the indirect subtraction methods. Additional studies are needed to determine which method allows for the most accurate measurement of flap thickness.

Fluorescent reporter transgenic mice for in vivo live imaging of angiogenesis and lymphangiogenesis.

The study of lymphangiogenesis is an emerging science that has revealed the lymphatic system as a central player in many pathological conditions including cancer metastasis, lymphedema, and organ graft rejection. A thorough understanding of the mechanisms of lymphatic growth will play a key role in the development of therapeutic strategies against these conditions. Despite the known potential of this field, the study of lymphatics has historically lagged behind that of hemangiogenesis. Until recently, significant strides in lymphatic studies were impeded by a lack of lymphatic-specific markers and suitable experimental models compared to those of the more immediately visible blood vasculature. Lymphangiogenesis has also been shown to be a key phenomenon in developmental biological processes, such as cell proliferation, guided migration, differentiation, and cell-to-cell communication, making lymphatic-specific visualization techniques highly desirable and desperately needed. Imaging modalities including immunohistochemistry and in situ hybridization are limited by the need to sacrifice animal models for tissue harvesting at every experimental time point. Moreover, the processes of mounting and staining harvested tissues may introduce artifacts that can confound results. These traditional methods for investigating lymphatic and blood vasculature are associated with several problems including animal variability (e.g., between mice) when replicating lymphatic growth environments and the cost concerns of prolonged, labor-intensive studies, all of which complicate the study of dynamic lymphatic processes. With the discovery of lymphatic-specific markers, researchers have been able to develop several lymphatic and blood vessel-specific, promoter-driven, fluorescent-reporter transgenic mice for visualization of lymphatics in vivo and in vitro. For instance, GFP, mOrange, tdTomato, and other fluorescent proteins can be expressed under control of a lymphatic-specific marker like Prospero-related homeobox 1 (Prox1), which is a highly conserved transcription factor for determining embryonic organogenesis in vertebrates that is implicated in lymphangiogenesis as well as several human cancers. Importantly, Prox1-null mouse embryos develop without lymphatic vessels. In human adults, Prox1 maintains lymphatic endothelial cells and upregulates proteins associated with lymphangiogenesis (e.g., VEGFR-3) and downregulates angiogenesis-associated gene expression (e.g., STAT6). To visualize lymphatic development in the context of angiogenesis, dual fluorescent-transgenic reporters, like Prox1-GFP/Flt1-DsRed mice, have been bred to characterize lymphatic and blood vessels simultaneously in vivo. In this review, we discuss the trends in lymphatic visualization and the potential usage of transgenic breeds in hemangiogenesis and lymphangiogenesis research to understand spatial and temporal correlations between vascular development and pathological progression.

Potential lymphangiogenesis therapies: Learning from current antiangiogenesis therapies-A review.

In recent years, lymphangiogenesis, the process of lymphatic vessel formation from existing lymph vessels, has been demonstrated to have a significant role in diverse pathologies, including cancer metastasis, organ graft rejection, and lymphedema. Our understanding of the mechanisms of lymphangiogenesis has advanced on the heels of studies demonstrating vascular endothelial growth factor C as a central pro-lymphangiogenic regulator and others identifying multiple lymphatic endothelial biomarkers. Despite these breakthroughs and a growing appreciation of the signaling events that govern the lymphangiogenic process, there are no FDA-approved drugs that target lymphangiogenesis. In this review, we reflect on the lessons available from the development of antiangiogenic therapies (26 FDA-approved drugs to date), review current lymphangiogenesis research including nanotechnology in therapeutic drug delivery and imaging, and discuss molecules in the lymphangiogenic pathway that are promising therapeutic targets.

Angiogenesis and lymphangiogenesis in corneal transplantation-A review.

Corneal transplantation has been proven effective for returning the gift of sight to those affected by corneal disorders such as opacity, injury, and infections that are a leading cause of blindness. Immune privilege plays an important role in the success of corneal transplantation procedures; however, immune rejection reactions do occur, and they, in conjunction with a shortage of corneal donor tissue, continue to pose major challenges. Corneal immune privilege is important to the success of corneal transplantation and closely related to the avascular nature of the cornea. Corneal avascularity may be disrupted by the processes of angiogenesis and lymphangiogenesis, and for this reason, these phenomena have been a focus of research in recent years. Through this research, therapies addressing certain rejection reactions related to angiogenesis have been developed and implemented. Corneal donor tissue shortages also have been addressed by the development of new materials to replace the human donor cornea. These advancements, along with other improvements in the corneal transplantation procedure, have contributed to an improved success rate for corneal transplantation. We summarize recent developments and improvements in corneal transplantation, including the current understanding of angiogenesis mechanisms, the anti-angiogenic and anti-lymphangiogenic factors identified to date, and the new materials being used. Additionally, we discuss future directions for research in corneal transplantation.

Prox1-GFP/Flt1-DsRed transgenic mice: an animal model for simultaneous live imaging of angiogenesis and lymphangiogenesis.

The roles of angiogenesis in development, health, and disease have been studied extensively; however, the studies related to lymphatic system are limited due to the difficulty in observing colorless lymphatic vessels. But recently, with the improved technique, the relative importance of the lymphatic system is just being revealed. We bred transgenic mice in which lymphatic endothelial cells express GFP (Prox1-GFP) with mice in which vascular endothelial cells express DsRed (Flt1-DsRed) to generate Prox1-GFP/Flt1-DsRed (PGFD) mice. The inherent fluorescence of blood and lymphatic vessels allows for direct visualization of blood and lymphatic vessels in various organs via confocal and two-photon microscopy and the formation, branching, and regression of both vessel types in the same live mouse cornea throughout an experimental time course. PGFD mice were bred with CDh5CreERT2 and VEGFR2lox knockout mice to examine specific knockouts. These studies showed a novel role for vascular endothelial cell VEGFR2 in regulating VEGFC-induced corneal lymphangiogenesis. Conditional deletion of vascular endothelial VEGFR2 abolished VEGFA- and VEGFC-induced corneal lymphangiogenesis. These results demonstrate the potential use of the PGFD mouse as a powerful animal model for studying angiogenesis and lymphangiogenesis.

TFOS DEWS II iatrogenic report.

Dry eye can be caused by a variety of iatrogenic interventions. The increasing number of patients looking for eye care or cosmetic procedures involving the eyes, together with a better understanding of the pathophysiological mechanisms of dry eye disease (DED), have led to the need for a specific report about iatrogenic dry eye within the TFOS DEWS II. Topical medications can cause DED due to their allergic, toxic and immuno-inflammatory effects on the ocular surface. Preservatives, such as benzalkonium chloride, may further aggravate DED. A variety of systemic drugs can also induce DED secondary to multiple mechanisms. Moreover, the use of contact lens induces or is associated with DED. However, one of the most emblematic situations is DED caused by surgical procedures such as corneal refractive surgery as in laser-assisted in situ keratomileusis (LASIK) and keratoplasty due to mechanisms intrinsic to the procedure (i.e. corneal nerve cutting) or even by the use of postoperative topical drugs. Cataract surgery, lid surgeries, botulinum toxin application and cosmetic procedures are also considered risk factors to iatrogenic DED, which can cause patient dissatisfaction, visual disturbance and poor surgical outcomes. This report also presents future directions to address iatrogenic DED, including the need for more in-depth epidemiological studies about the risk factors, development of less toxic medications and preservatives, as well as new techniques for less invasive eye surgeries. Novel research into detection of early dry eye prior to surgeries, efforts to establish appropriate therapeutics and a greater attempt to regulate and oversee medications, preservatives and procedures should be considered.

Risk profiles of ectasia after keratorefractive surgery.

PURPOSE OF REVIEW: To identify and evaluate the risk factors of iatrogenic ectasia after refractive surgery. RECENT FINDINGS: We reviewed recently published papers that identified various risk factors associated with ectasia after LASIK, photorefractive keratectomy, small incision lenticule extraction, and other refractive surgical procedures. We also attempted to evaluate the relative contributions of these factors to the development of ectasia following refractive surgery. Forme fruste keratoconus, genetic predisposition to keratoconus, low residual stromal bed thickness (through high myopia, thin preoperative cornea, or thick LASIK flap), and irregular corneal topography have been identified as risk factors for keratectasia development after refractive surgical procedures. A newly proposed metric, percentage tissue altered, has been reported to be a robust indicator for post LASIK ectasia risk calculation. Several cases of keratectasia have also been reported 6 to 12 months following minimally invasive small incision lenticule extraction procedure. Other risk factors associated with iatrogenic ectasia include eye rubbing, young age, and pregnancy. SUMMARY: Ectasia after refractive surgery is a relatively rare complication which can lead to sight-threatening complications if not detected and treated in time. It is important to continue our quest to improve our methods of identifying absolute and relative risk factors of ectasia and their cut-off values following various keratorefractive surgical procedures.

Potential role of corneal epithelial cell-derived exosomes in corneal wound healing and neovascularization.

Specific factors from the corneal epithelium underlying the stimulation of stromal fibrosis and myofibroblast formation in corneal wound healing have not been fully elucidated. Given that exosomes are known to transfer bioactive molecules among cells and play crucial roles in wound healing, angiogenesis, and cancer, we hypothesized that corneal epithelial cell-derived exosomes may gain access to the underlying stromal fibroblasts upon disruption of the epithelial basement membrane and that they induce signaling events essential for corneal wound healing. In the present study, exosome-like vesicles were observed between corneal epithelial cells and the stroma during wound healing after corneal epithelial debridement. These vesicles were also found in the stroma following anterior stromal keratectomy, in which surgical removal of the epithelium, basement membrane, and anterior stroma was performed. Exosomes secreted by mouse corneal epithelial cells were found to fuse to keratocytes in vitro and to induce myofibroblast transformation. In addition, epithelial cell-derived exosomes induced endothelial cell proliferation and ex vivo aortic ring sprouting. Our results indicate that epithelial cell-derived exosomes mediate communication between corneal epithelial cells and corneal keratocytes as well as vascular endothelial cells. These findings demonstrate that epithelial-derived exosomes may be involved in corneal wound healing and neovascularization, and thus, may serve as targets for potential therapeutic interventions.

Proangiogenic Interactions of Vascular Endothelial MMP14 With VEGF Receptor 1 in VEGFA-Mediated Corneal Angiogenesis.

PURPOSE: Matrix metalloproteinase 14 (MMP14) has been shown to be required for corneal angiogenesis. We hypothesized that the proangiogenic activity of MMP14 may be based on its selective binding to, and cleaving of, vascular endothelial growth factor receptor 1 (VEGFR1), but not VEGFR2 or VEGFR3. METHODS: Recombinant human (rh)VEGFR1, R2, and R3 were incubated with human MMP14, and the reaction mixtures were analyzed by SDS-PAGE and Coomassie blue staining. Surface plasmon resonance was used to determine the equilibrium constants (KD) for binding between MMP14 and VEGFA versus rhVEGFR1, R2, and R3. Extracellular signal-regulated kinase (ERK) phosphorylation was assayed in vascular endothelial cells after incubation with VEGF and various concentrations of MMP14. Ex vivo aortic ring tube formation assays and VEGFA micropocket corneal neovascularization assays were performed using Flk1Cre/Flk1mCherry/MMP14lox and Flk1mCherry/MMP14lox control mice. RESULTS: Maxtrix metalloproteinase 14 increased VEGFA-induced ERK phosphorylation in a time- and concentration-dependent manner in vascular endothelial cells. Aortic ring assays showed diminished vessel sprouting in vitro in response to VEGFA, but not to basic fibroblast growth factor, in mice with conditional deletion of vascular MMP14 (Flk1creMMP14lox) compared with that in MMP14lox control mice. In addition, diminished VEGFA-induced corneal angiogenesis was seen in flk1creMMP14lox mice compared with MMP14lox mice in vivo. CONCLUSIONS: Our findings indicate that VEGFR1 interaction with MMP14 and the enzymatic activity of MMP14 are necessary for VEGFA-induced angiogenesis. Additionally, selective cleavage of VEGFR1 by MMP14 may play an important role in VEGFA-induced corneal angiogenesis.