Purpose. Lactate, a key metabolite in the retinal tissue, has been implicated in regulating retinal blood flow to match retinal metabolic demand. However, the direct effect of lactate on retinal vascular tone and the possible underlying signaling mechanisms remain unknown. In the present study, the roles of endothelium-derived vasodilators, guanylyl cyclase, and potassium channels were examined in lactate-induced dilation of retinal arterioles in vitro.

Development of therapeutic approaches to treat vascular dysfunction and thrombosis at disease- and patient-specific levels is an exciting proposed direction in biomedical research. However, this cannot be achieved with animal preclinical models alone, and new in vitro techniques, like human organ-on-chips, currently lack inclusion of easily obtainable and phenotypically-similar human cell sources. Therefore, there is an unmet need to identify sources of patient primary cells and apply them in organ-on-chips to increase personalized mechanistic understanding of diseases and to assess drugs. In this study, we provide a proof-of-feasibility of utilizing blood outgrowth endothelial cells (BOECs) as a disease-specific primary cell source to analyze vascular inflammation and thrombosis in vascular organ-chips or “vessel-chips”. These blood-derived BOECs express several factors that confirm their endothelial identity. The vessel-chips are cultured with BOECs from healthy or diabetic patients and form an intact 3D endothelial lumen. Inflammation of the BOEC endothelium with exogenous cytokines reveals vascular dysfunction and thrombosis in vitro similar to in vivo observations. Interestingly, our study with vessel-chips also reveals that unstimulated BOECs of type 1 diabetic pigs show phenotypic behavior of the disease – high vascular dysfunction and thrombogenicity – when compared to control BOECs or normal primary endothelial cells. These results demonstrate the potential of organ-on-chips made from autologous endothelial cells obtained from blood in modeling vascular pathologies and therapeutic outcomes at a disease and patient-specific level.

Purpose.: Although the arteriolar segment contributes to flow regulation, there is sparse information at the single microvessel level on how vasomotor function is regulated in the human retina. The authors have previously reported vasoreactivity and its underlying mechanisms in isolated porcine retinal arterioles. Herein, they studied human retinal arterioles for comparison. Methods.: Retinal tissues were obtained from seven patients undergoing enucleation. Human and porcine retinal arterioles were isolated and pressurized to 55 cm H₂O luminal pressure for vasoreactivity study using videomicroscopic techniques. Results.: Isolated human and porcine retinal arterioles developed myogenic tone and dilated dose dependently to bradykinin, adenosine, and sodium nitroprusside. Stepwise increases in luminal flow produced graded dilation with approximately 60% dilation at the highest flow tested. Nitric oxide (NO) synthase inhibitor L-NAME nearly abolished dilations to bradykinin and flow and attenuated the adenosine-induced dilation without altering the response to nitroprusside. Endothelin-1 caused dose-dependent constriction. Rho kinase (ROCK) inhibitor H-1152 blocked both myogenic tone and endothelin-1–induced constriction. Responses of retinal arterioles to all agonists and increased flow were similar between pigs and humans. Conclusions.: Isolated human retinal arterioles dilate to bradykinin and increased flow in an NO-dependent manner. NO contributes, in part, to adenosine-induced vasodilation. Conversely, ROCK activation mediates myogenic tone and endothelin-1–induced vasoconstriction. Similarities in these vasoactive responses and the underlying mechanisms between human and porcine retinal arterioles support the latter as a viable experimental model of the human retinal microcirculation.

Purpose. Adenosine is a potent vasodilator of retinal microvessels and is implicated to be a major regulator of retinal blood flow during metabolic stress. However, the receptor subtypes and the underlying signaling mechanism responsible for the dilation of retinal microvessels in response to adenosine remain unclear. In the present study, the roles of specific adenosine receptor subtypes, nitric oxide (NO), and adenosine triphosphate (ATP)–sensitive K⁺ (K_ATP) channels in adenosine-induced dilation of retinal arterioles in vitro were examined.

Purpose. Activation of the endothelin (ET) system has been implicated in the pathogenesis of retinal ischemic disease. Although ET-1, the predominant endogenous isoform of ET, has been shown to cause constriction of retinal vessels, the expression and functional significance of its synthesis and the involved specific ET receptors in retinal arterioles remain unknown. The authors examined the roles of ET_A and ET_B receptors and of endothelin-converting enzyme (ECE)-1 in ET-1–induced vasomotor responses of single retinal arterioles. methods. To exclude systemic confounding effects, porcine retinal arterioles were isolated for vasoreactivity and molecular studies. results. Isolated and pressurized retinal arterioles developed basal tone and constricted in a manner dependent on concentration to ET-1. ET-1 precursor big ET-1 elicited time-dependent vasoconstriction over 20 minutes, which was blocked by the ECE-1 inhibitor phosphoramidon. ET_A receptor antagonist BQ123 inhibited most (approximately 90%) of vasoconstrictions to ET-1 and big ET-1. ET_B receptor agonist sarafotoxin also elicited concentration-dependent constriction of retinal arterioles but with significantly less potency than ET-1. ET_B receptor antagonist BQ788 abolished vasoconstriction to sarafotoxin but only slightly reduced responses to ET-1 and big ET-1. Protein and mRNA expressions of ET_A, ET_B, and ECE-1 were detected in retinal arterioles. Immunohistochemistry revealed ET_A and ET_B receptors predominantly in smooth muscle and ECE-1 predominantly in endothelium and smooth muscle. conclusions. ET-1 elicits constriction of retinal arterioles predominantly through the activation of smooth muscle ET_A receptors. Endogenous production of ET-1 from vascular ECE-1 is sufficient to evoke ET_A receptor–dependent constriction in retinal arterioles.

Purpose: Endothelin-1 (ET-1) is a potent vasoconstrictor peptide implicated in retinal venous pathologies such as diabetic retinopathy and retinal vein occlusion. However, underlying mechanisms contributing to venular constriction remain unknown. Thus, we examined the roles of ET-1 receptors, extracellular calcium (Ca²⁺), L-type voltage-operated calcium channels (L-VOCCs), Rho kinase (ROCK), and protein kinase C (PKC) in ET-1-induced constriction of retinal venules. Methods: Porcine retinal venules were isolated and pressurized for vasoreactivity study using videomicroscopic techniques. Protein and mRNA were analyzed using molecular tools. Results: Retinal venules developed basal tone and constricted concentration-dependently to ET-1. The ET_A receptor (ET_AR) antagonist BQ123 abolished venular constriction to ET-1, but ET_B receptor (ET_BR) antagonist BQ788 had no effect on vasoconstriction. The ET_BR agonist sarafotoxin S6c did not elicit vasomotor activity. In the absence of extracellular Ca²⁺, venules lost basal tone and ET-1–induced constriction was nearly abolished. Although L-VOCC inhibitor nifedipine also reduced basal tone and blocked vasoconstriction to L-VOCC activator Bay K8644, constriction of venules to ET-1 remained. The ROCK inhibitor H-1152 but not PKC inhibitor Gö 6983 prevented ET-1-induced vasoconstriction. Protein and mRNA expressions of ET_ARs and ET_BRs, along with ROCK1 and ROCK2 isoforms, were detected in retinal venules. Conclusions: Extracellular Ca²⁺ entry via L-VOCCs is essential for developing and maintaining basal tone of porcine retinal venules. ET-1 causes significant constriction of retinal venules by activating ET_ARs and extracellular Ca²⁺ entry independent of L-VOCCs. Activation of ROCK signaling, without involvement of PKC, appears to mediate venular constriction to ET-1 in the porcine retina.