Travis W. Hein

Background/Objectives: The correlation between in vivo morphological and functional changes in the degenerating retina in a large animal model of retinitis pigmentosa (RP) has not been characterized longitudinally. Herein, spectral domain optical coherence tomography (SD-OCT) was used to monitor the dynamic morphological changes in the Pro23His rhodopsin transgenic (TgP23H) pig model of RP and was correlated with electroretinography (ERG) in the rapid, early phase of photoreceptor degeneration. Methods: TgP23H and wild-type (WT) hybrid pig littermates at the ages of postnatal days 30 (P30), P60, and P90 were studied. The thickness of different retinal layers was quantified using SD-OCT and compared with histology. Retinal function was evaluated with ERG at corresponding time points. Results: In the WT pigs, retinal morphology on SD-OCT was consistent throughout the observation period. In the TgP23H pigs, the retinal thickness decreased significantly from P30 to P90. Moreover, the relative intensity of the ellipsoid zone (EZ) progressively decreased, while the intensity of the interdigitation zone–retinal pigment epithelium (IZ-RPE) progressively increased during this period. Morphological changes in SD-OCT corresponded with histology, as well as the progressively decreased amplitude of the ERG photopic a- and b-waves in the TgP23H pigs. Conclusions: Retinal degeneration can be quantified using SD-OCT by measuring retinal thickness and the intensity of the EZ and IZ-RPE bands in the TgP23H pig. The SD-OCT results correspond with the histologic and ERG assessments of retinal degeneration. These data provide a foundation for future preclinical studies investigating potential new therapeutic strategies in a large animal model of retinitis pigmentosa.

Correlation of in vivo morphological and functional changes in the degenerating retina in a large animal model of retinitis pigmentosa (RP) has not been characterized longitudinally. Herein, spectral domain optical coherence tomography (SD-OCT) was used to monitor the dynamic morphological changes in the Pro23His rhodopsin transgenic (TgP23H) pig model of RP and was correlated with electroretinography (ERG) in the rapid, early phase of photoreceptor degenera-tion. TgP23H and wild type (Wt) hybrid pig littermates at the ages of P30, P60, and P90 were studied. The thickness of different retinal layers was quantified using SD-OCT and compared with histology. Retinal function was evaluated with ERG at corresponding time points. In the Wt pig, retinal morphology on SD-OCT was consistent throughout the observation period. In the TgP23H pig, the retinal thickness decreased significantly from P30 to P90. Moreover, the relative intensity of the ellipsoid zone (EZ) progressively decreased, while the intensity of the interdigita-tion zone-retinal pigment epithelium (IZ-RPE) progressively increased during this period. Mor-phological changes in SD-OCT corresponded with histology, as well as the progressively de-creased amplitude of the ERG photopic a- and b-waves in TgP23H pigs. Thus, retinal degenera-tion can be quantified using SD-OCT by measuring retinal thickness and the intensity of the EZ and IZ-RPE bands in the TgP23H pig. The SD-OCT results correspond with the histologic and ERG assessments of retinal degeneration. These data provide a foundation for future preclinical studies investigating potential new therapeutic strategies in a large animal model of retinitis pigmentosa.

Metabolic stress on the heart can cause dilation of coronary arterioles for blood flow recruitment. Although potassium ions (K⁺) released from the myocardium are a major mediator for this response, the underlying signaling pathways for vasodilation are incompletely understood. Herein, the roles of smooth muscle inward-rectifier K⁺ channel subtype 2.1 (K_ir2.1) and Na⁺/K⁺-ATPase were examined. Porcine coronary arterioles were isolated, cannulated, and pressurized for vasomotor study. Vessels developed basal tone and dilated concentration-dependently to extraluminal K⁺ from 7 to 20 mM. Higher K⁺ concentrations (25–40 mM) caused graded vasoconstriction. Vasodilation to K⁺ (10 mM) was not altered by endothelial removal, and blockade of ATP-sensitive K⁺ channels, voltage-sensitive K⁺ channels, or calcium-activated K⁺ channels did not affect K⁺-induced vasodilation. However, sustained but not abrupt transient vasodilation to K⁺ was reduced by the nonspecific K_ir channel inhibitor Ba²⁺ or K_ir2.1 channel blocker chloroethylclonidine. The Na⁺/K⁺-ATPase inhibitor ouabain attenuated K⁺-elicited vasodilation, and ouabain with Ba²⁺ abolished the response. Transfection of arterioles with K_ir2.1 antisense oligonucleotides abolished sustained but not transient dilation. It is concluded that extraluminal K⁺ elevation within the physiological range induces initial transient dilation of porcine coronary arterioles by activating smooth muscle Na⁺/K⁺-ATPase and sustained dilation via smooth muscle K_ir2.1 channels.

Metabolic stress on the heart can cause dilation of coronary arterioles for blood flow recruitment. Although potassium ions (K+) released from the myocardium are a major mediator for this response, the underlying signaling pathways for vasodilation are incompletely understood. Herein, roles of smooth muscle inward-rectifier K+ channel subtype 2.1 (Kir2.1) and Na+/K+ ATPase were examined. Porcine coronary arterioles were isolated, cannulated, and pressurized for vasomotor study. Vessels developed basal tone and dilated concentration-dependently to extraluminal K+ from 7 to 20 mM. Higher K+ concentrations (25-40 mM) caused graded vasoconstriction. Vasodilation to K+ (10 mM) was not altered by endothelial removal, and blockade of ATP-sensitive K+ channels, voltage-sensitive K+ channels, or calcium-activated K+ channels did not affect K+-induced vasodilation. However, sustained but not abrupt transient vasodilation to K+ was reduced by nonspecific Kir channel inhibitor Ba2+ or specific Kir2.1 channel blocker chloroethylclonidine. The Na+/K+ ATPase inhibitor ouabain attenuated K+-elicited vasodilation, and ouabain with Ba2+ abolished the response. Transfection of arterioles with Kir2.1 antisense oligonucleotides abolished sustained but not transient dilation. It is concluded that extraluminal K+ elevation within the physiological range induces initial transient dilation of porcine coronary arterioles by activating Na+/K+ ATPase and sustained dilation via smooth muscle Kir2.1 channels.

In diabetes, the enzyme arginase is upregulated, which may compete with endothelial nitric oxide (NO) synthase (eNOS) for their common substrate L-arginine and compromise NO-mediated vasodilation. However, this eNOS uncoupling can lead to superoxide production and possibly vasodilator hydrogen peroxide (H₂O₂) formation to compensate for NO deficiency. This hypothesis was tested in coronary arterioles isolated from pigs with 2-week diabetes after streptozocin injection. The NO-mediated vasodilation induced by flow and VEGF was abolished by NOS inhibitor L-NAME and phosphoinositide 3-kinase (PI3K) inhibitor wortmannin but was not affected by arginase inhibitor N^ω-hydroxy-nor-L-arginine (nor-NOHA) or H₂O₂ scavenger catalase in control pigs. With diabetes, this vasodilation was partially blunted, and the remaining vasodilation was abolished by catalase and wortmannin. Administration of L-arginine or nor-NOHA restored flow-induced vasodilation in an L-NAME sensitive manner. Diabetes did not alter vascular superoxide dismutase 1, catalase, and glutathione peroxidase mRNA levels. This study demonstrates that endothelium-dependent NO-mediated coronary arteriolar dilation is partially compromised in early type 1 diabetes by reducing eNOS substrate L-arginine via arginase activation. It appears that upregulated arginase contributes to endothelial NO deficiency in early diabetes, but production of H₂O₂ during PI3K-linked eNOS uncoupling likely compensates for and masks this disturbance.

Protein kinase C (PKC) activation can evoke vasoconstriction and contribute to coronary disease. However, it is unclear whether PKC activation, without activating the contractile machinery, can lead to coronary arteriolar dysfunction. The vasoconstriction induced by the PKC activator phorbol 12,13-dibutyrate (PDBu) was examined in isolated porcine coronary arterioles. The PDBu-evoked vasoconstriction was sensitive to a broad-spectrum PKC inhibitor but not affected by inhibiting PKCβ2 or Rho kinase. After exposure of the vessels to a sub-vasomotor concentration of PDBu (1 nmol/L, 60 min), the endothelium-dependent nitric oxide (NO)-mediated dilations in response to serotonin and adenosine were compromised but the dilation induced by the NO donor sodium nitroprusside was unaltered. PDBu elevated superoxide production, which was blocked by the superoxide scavenger Tempol. The impaired NO-mediated vasodilations were reversed by Tempol or inhibition of PKCβ2, xanthine oxidase, c-Jun N-terminal kinase (JNK) and Rho kinase but were not affected by a hydrogen peroxide scavenger or inhibitors of NAD(P)H oxidase and p38 kinase. The PKCβ2 protein was detected in the arteriolar wall and co-localized with endothelial NO synthase. In conclusion, activation of PKCβ2 appears to compromise NO-mediated vasodilation via Rho kinase-mediated JNK signaling and superoxide production from xanthine oxidase, independent of the activation of the smooth muscle contractile machinery.

Diabetes elevates endothelin-1 (ET-1) in the vitreous and enhances constriction of retinal venules to this peptide. However, mechanisms contributing to ET-1-induced constriction of retinal venules are incompletely understood. We examined roles of sodium-hydrogen exchanger-1 (NHE1), protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), and extracellular calcium (Ca²⁺) in retinal venular constriction to ET-1 and the impact of diabetes on these signaling molecules. Retinal venules were isolated from control and streptozocin-induced diabetic pigs for in vitro studies. ET-1-induced vasoconstriction was abolished in the absence of extracellular Ca²⁺ and sensitive to c-Jun N-terminal kinase (JNK) inhibitor SP600125 but unaffected by extracellular signal-regulated kinase (ERK) inhibitor PD98059, p38 kinase inhibitor SB203580, or a broad-spectrum PKC inhibitor Gö 6983. Diabetes (2 weeks) enhanced venular constriction to ET-1, which was insensitive to PD98059 and Gö 6983 but was prevented by NHE1 inhibitor cariporide, SB203580, and SP600125. In conclusion, extracellular Ca²⁺ entry and activation of JNK, independent of ERK and PKC, mediate constriction of retinal venules to ET-1. Diabetes activates p38 MAPK and NHE1, which cause enhanced venular constriction to ET-1. Treatments targeting these vascular molecules may lessen retinal complications in early diabetes.

Purpose: Endothelin-1 (ET-1) is a potent vasoactive factor implicated in development of diabetic retinopathy, which is commonly associated with retinal edema and hyperglycemia. Although the vasomotor activity of venules contributes to the regulation of tissue fluid homeostasis, responses of human retinal venules to ET-1 under euglycemia and hyperglycemia remain unknown and the ET-1 receptor subtype corresponding to vasomotor function has not been determined. Herein, we addressed these issues by examining the reactivity of isolated human retinal venules to ET-1, and results from porcine retinal venules were compared. Methods: Retinal tissues were obtained from patients undergoing enucleation. Human and porcine retinal venules were isolated and pressurized to assess diameter changes in response to ET-1 after exposure to 5 mM control glucose or 25 mM high glucose for 2 hours. Results: Both human and porcine retinal venules exposed to control glucose developed similar basal tone and constricted comparably to ET-1 in a concentration-dependent manner. ET-1–induced constrictions of human and porcine retinal venules were abolished by ET_A receptor antagonist BQ123. During high glucose exposure, basal tone of human and porcine retinal venules was unaltered but ET-1–induced vasoconstrictions were enhanced. Conclusions: ET-1 elicits comparable constriction of human and porcine retinal venules by activation of ET_A receptors. In vitro hyperglycemia augments human and porcine retinal venular responses to ET-1. Translational Relevance: Similarities in vasoconstriction to ET-1 between human and porcine retinal venules support the latter as an effective model of the human retinal microcirculation to help identify vascular targets for the treatment of retinal complications in patients with diabetes.

Purpose: Retinal vasomotor activity can be regulated by two major endothelial enzymes, nitric oxide synthase (NOS) and cyclooxygenase (COX). The vascular arginase also consumes a NOS substrate and thus impedes NOS-mediated vasodilation. Diabetes mellitus exhibits vascular complications in the retina with elevated oxidative stress and compromised NOS-mediated vasodilation. However, the underlying molecular mechanisms remain unclear, and the effect of diabetes on COX-mediated vasodilation is unknown. Herein, we examined the relative impact of diabetes on retinal arteriolar dilations to COX and NOS activation and the roles of arginase and superoxide in diabetes-induced vasomotor dysfunction. Methods: Retinal arterioles were isolated from streptozocin-induced diabetic pigs (2 weeks of hyperglycemia, 433 ± 27 mg/dL) or age-matched control pigs (97 ± 4 mg/dL). The vasodilations to bradykinin (NOS activator) and histamine (NOS/COX activator) were examined in vitro. Results: Retinal arteriolar dilations to histamine and bradykinin were significantly reduced after 2 weeks of diabetes. The NOS inhibitor N^G-nitro-L-arginine methyl ester (L-NAME) attenuated the dilations of control vessels, but not diabetic vessels, to histamine. In the presence of L-NAME and COX inhibitor indomethacin, histamine-induced dilations of control and diabetic vessels were reduced similarly. Treatment of diabetic vessels with arginase inhibitor nor-NOHA, but not superoxide dismutase mimetic TEMPOL, preserved both histamine- and bradykinin-induced dilations in an L-NAME-sensitive manner. Conclusions: Arginase, rather than superoxide, impairs endothelium-dependent NOS-mediated dilation of retinal arterioles during diabetes, whereas vasodilation mediated by COX remains intact. Blockade of vascular arginase may improve endothelial function of retinal arterioles during early onset of diabetes.

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.