Diabetic retinopathy (DR), the most common microvascular complication of diabetes (DM), is the leading cause of blindness in working-age individuals. Fortunately, manifestation of DR is not coincident with the onset of DM. It takes over two decades for patients to develop sight-threatening DR. While the mechanism of resilience to diabetic retinopathy (RDR) is unknown, it is likely to involve suppression of DM-driven events that damage the retina. For instance, DM damages the retina and causes DR by accelerating senescence of the retinal vasculature. Since mitochondrial dysfunction drives senescence, we posit that RDR involves enforcing mitochondrial functionality in the face of DM. Our recent discoveries support this hypothesis (Fig 2). In an in vitro model, increased clearance of dysfunctional mitochondria (mitophagy) is associated with acquisition of RDR and required for cells to retain key features of RDR. Furthermore, mitophagy increases in the retinal vessels of mice that have acquired RDR. As mice lose RDR mitophagy declines. These in vitro and in vivo data support our working hypothesis that RDR protects from DM-driven senescence by increasing mitophagy. Ongoing studies are designed to further test this hypothesis and develop approaches to prevent the loss of RDR, which will indefinitely delay the onset of DR.

Anti-vascular endothelial growth factor (VEGF) treatment is used in patients to control blood vessel permeability and angiogenesis. The response of patients to such therapy has revealed that anti-VEGF can also promote or inhibit fibrosis, i.e., the nature of its effect is context dependent (Figure 1). The overall goal of this project is to unravel the mechanism by which anti-VEGF prevents and promotes fibrosis in the context of proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR), respectively.

Pathogenesis of proliferative vitreoretinopathy (PVR) includes enduring activation of the platelet-derived growth factor (PDGF) receptor alpha (PDGFRa), which enforces the viability of mesenchymal cells that have been displaced into the vitreous. Such cells undergo EMT, proliferate, produce an epiretinal membrane on or within the retina and contract it, thereby causing retinal detachment. Anti-VEGF protects experimental animals from developing PVR by preventing durable activation of PDGFRa, and thereby triggers apoptosis of mesenchymal cells.  While these studies begin to explain how anti-VEGF protects from PVR, they appear to underestimate the beneficial effect of anti-VEGF. Chronic activation of PDGFRa also drives EMT/fibrosis. Consequently, we posit that the beneficial effect of anti-VEGF includes inhibition of fibrosis in the context of PVR. However, in contrast with anti-VEGF’s putative ability to inhibit fibrosis in PVR, it is profibrotic in the context of PDR. In the PDR setting, anti-VEGF’s target is endothelial cells, which can undergo EndMT (endothelial mesenchymal transition). We are also investigating the mechanism by which anti-VEGF promotes fibrosis in the context of PDR.

The therapeutic efficacy of anti-VEGF (vascular endothelial growth factor) in patients with diabetic retinopathy (DR) demonstrates the central role of VEGF in the pathogenesis of this vascular disease. Not all patients experience adequate benefit from anti-VEGF, and even those who do, can suffer detrimental side effects of this therapeutic option. The overall goal of this project is to identify new therapeutic approaches and targets for patients who have developed vision threatening forms of diabetic retinopathy such as proliferative diabetic retinopathy (PDR) diabetic macular edema (DME).

The standard of care for patients that have advanced to end-stage PDR is surgery to remove pathological blood vessels. The availability of such clinical specimens constitutes an opportunity to investigate the transcriptional profile within pathological blood vessels in patients. We embraced this opportunity by assembling a team to consent patients, collect surgical specimens, isolate the CD31+ cells from the pathological vessels, and determine their gene expression profile using RNA sequencing. To date, we generated high-quality PDR patient endothelial transcriptomes from the CD31+ cells from ten end-stage PDR patients. This project involves mining these transcriptomes to define the molecular signature of PDR within the endothelium. The molecular signature will then be used to establish novel classes of therapeutic targets, identify alternative targets for anti-VEGF therapy, and generate pre-clinical data for the best targets.

The discovery of kinases that phosphorylate proteins on tyrosine residues and their association with proliferation of cells captured the attention of both basic and translational researchers. As a postdoctoral fellow in Jonathan Cooper’s lab I focused on the receptor for platelet-derived growth factor (PDGF). I discovered that tyrosine autophosphorylation of the PDGFR not only enhanced this receptor’s intrinsic kinase activity, but created docking sites for SH2 domain-containing proteins. The PDGFR autophosphorylates at multiple tyrosine residues, which, together with the surrounding amino acids, constitute a binding site for a specific a SH2 domain-containing protein. Other groups focusing on different receptor tyrosine kinases came to similar conclusions. These studies contributed to the realization that tyrosine phosphorylation of proteins governs protein-protein interaction.

_{Kazlauskas A, Cooper JA. Autophosphorylation of the PDGF receptor in the kinase insert region regulates interactions with cell proteins. Cell 1989; 58:1121-33.
Kazlauskas A, Ellis C, Pawson T, Cooper JA. Binding of GAP to activated PDGF receptors. Science 1990; 247:1578-81.
Kazlauskas A, Cooper JA. Phosphorylation of the PDGF receptor β subunit creates a tight binding site for phosphatidylinositol 3 kinase. EMBO J 1990; 9:3279-86.
Valius M, Bazenet C, Kazlauskas A. Tyrosine 1021 and 1009 are phosphorylation sites in the carboxyterminus of the platelet-derived growth factor receptor β subunit and are required for binding of phospholipase Cγ and a 64 kd protein, respectively. Mol Cell Biol 1993; 13:133-43.}

One of the key scientific questions at this point in time was how growth factors trigger cellular responses. The realization that SH2 domain-containing proteins that were being recruited to the activated PDGFR were signaling enzymes, and that their association with PDGFR often activated them, led to the hypothesis that such signaling enzymes were triggering signaling pathways that directed various cellular responses. Understanding how these signaling enzymes were being activated by growth factor receptors enabled the generation of reagents to selectively prevent the activation of a given signaling enzyme. These tools included PDGFR phosphorylation site mutants that selectively failed to engage a desired signaling enzyme. Working with a number of collaborators we defined the signaling enzymes that were essential for growth factor-driven proliferation and migration of cells. This information guided subsequent efforts to identify additional members of relevant signaling pathways, e.g. enzymes such as Akt, which acted downstream of phosphatidylinositol 3-kinase (PI3K).

_{Valius M, Kazlauskas A. Phospholipase Cγ and phosphatidylinositol 3 kinase are the downstream mediators of the PDGF receptor’s mitogenic signal. Cell 1993; 73:321-34.
Kundra V, Escobedo J, Kazlauskas A, Williams LT, Zetter B. Regulation of chemotaxis by the platelet-derived growth factor receptor-b. Nature 1994; 367:474-6.
Franke TF, Yang S-I, Chan TO, Datta K, Kazlauskas A, Morrison DK, Kaplan DR, Tsichlis PN. The protein kinase encoded by the Akt proto-oncogene is a target of the platelet-derived growth factor (PDGF)-activated phosphatidylinositol 3-kinase (PI 3-kinase). Cell 1995; 81:727-36.
Klinghoffer RA, Duckworth B, Valius M, Cantley L, Kazlauskas A. Platelet-derived growth factor-dependent activation of phosphatidylinositol 3-kinase is regulated by receptor binding of SH2-domain-containing proteins which influence Ras activity. Mol Cell Biol 1996; 16:5905-14.}

At this point in time it was known that growth factors were capable of driving cells through the cell cycle, and that their input was required only for part of the cycle, namely, to move cells through the restriction point, which was close to the G1/S boundary. Which signaling enzymes were necessary, and when they contributed during this roughly 10-12 hr period was largely unknown. We discovered that growth factor-driven cell cycle progression did not require continuous signaling. Rather, growth factor-induced signaling was discontinuous, and appropriately timed discontinuous signaling was sufficient for progression through the cell cycle. We also identified which signaling enzymes were required and when they contributed. These discoveries guided subsequent investigation by other groups who defined the existence and timing of additional gating mechanisms for growth factor-driven cell cycle progression.

_{Jones SM, Klinghoffer R, Prestwich GD, Toker A, Kazlauskas A. PDGF induces an early and a late wave of PI 3-kinase activity, and only the late wave is required for progression through G1. Cur Biol 1999; 9:512-21.
Balciunaite E, Jones S, Toker A, Kazlauskas A. PDGF initiates two distinct phases of protein kinase C activity that make unequal contributions to the G0 to S transition. Cur Biol 2000; 10:261-7.
Jones SM, Kazlauskas A. Growth factor-dependent mitogenesis requires two distinct phases of signalling. Nature Cell Biol 2001; 3:165-72.
Balciunaite E, Kazlauskas A. Early phosphoinositide 3-kinase activity is required for late activation of protein kinase C epsilon in platelet-derived growth factor-stimulated cells: evidence for signaling across a large temporal gap. Biochem J 2001; 358:281-5.}

Focusing on the fate of newly formed blood vessels, we discovered that they are instructed to regress by the bioactive phospholipid LPA (lysophosphatidic acid). Since LPA is ubiquitous, how do newly formed blood vessels persist? The reason in part is because pro-angiogenic growth factors such as IGF-1 persistently activate signaling pathways that overcome LPA-directed regression. These concepts begin to explain why there are some many growth factors that participate in angiogenesis. Agents like VEGF trigger the formation of new blood vessel, while IGF-1 allows them to persist by overcoming the action of regression factors such as LPA.

These insights provided the foundation to investigate how diabetes disrupts angiogenic homeostasis. The high glucose aspect of diabetes rewires the signaling network in endothelial cells such that they become resistant to LPA-driven regression. This discovery begins to explain why pathological blood vessels accumulate in vitreous of patients with PDR (proliferative diabetic retinopathy), despite the presence of sufficient quantities of LPA to trigger their regression. Our contribution in this area has been both identification of signaling events that govern angiogenic homeostasis, and how diabetes rewires this network in endothelial cells.

_{Im E, Motiejunaite R, Aranda J, Park EY, Federico L, Kim TI, Clair T, Stracke ML, Smyth S, Kazlauskas A. Phospholipase Cgamma activation drives increased production of autotaxin in endothelial cells and lysophosphatidic acid-dependent regression. Mol Cell Biol 2010; 30:2401-10.
Aranda J, Motiejunaite R, Im E, Kazlauskas A. Diabetes Disrupts the Response of Retinal Endothelial Cells to the Angiomodulator Lysophosphatidic Acid. Diabetes 2012; 61:1225-33.
Motiejūnaitė R, Aranda J, Kazlauskas A. Pericytes prevent regression of endothelial cell tubes by accelerating metabolism of lysophosphatidic acid. Microvasc Res. 2014; 93:62-71.
Jacobo SM, Kazlauskas A. Insulin-like growth factor 1 (IGF-1) stabilizes nascent blood vessels. J Biol Chem. 2015; 290:6349-60.}

My most translational contribution to science has been in the area of a blinding disease called proliferative vitreoretinopathy (PVR). Patients who undergo surgery to correct a detached retina can develop this condition, which is difficult to manage. PVR develops when cells that are displaced into the vitreous proliferate, produce extracellular matrix and assemble it into an epiretinal membrane. Contraction of the epiretinal membrane detaches the retina and mandates additional episodes of surgery to re-attach the retina. We discovered that growth factors in the vitreous enable the survival of the misplaced cells. Furthermore, while there is a plethora of vitreal growth factors, their contribution to pathogenesis depends on their ability to indirectly activate PDGFRa, which is a novel mode of activation that promotes viability of displaced cells. Surprisingly, some of the growth factors antagonize the ability of other growth factors to indirectly activate PDGFRa. The discovery of both the hierarchy amongst vitreal growth factors, and the essential signaling events downstream of indirectly activated PDGFRa guided development of approaches to prevent experimental PVR.

_{Andrews A, Balciunaite E, Leong FL, Tallquist M, Soriano P, Refojo M, Kazlauskas A. Platelet-derived growth factor plays a key role in proliferative vitreoretinopathy. Invest Ophthalmol Vis Sci 1999; 40:2683-9.
Lei H, Velez G, Kazlauskas A. Pathological signaling via PDGFR{alpha} involves chronic activation of Akt and suppression of p53. Mol Cell Biol 2011; 31:1788-99.
Lei H, Kazlauskas A. A reactive oxygen species-mediated, self-perpetuating loop persistently activates platelet-derived growth factor receptor α. Mol Cell Biol. 2014; 34:110-22.
 Pennock S, Rheaume MA, Mukai S, Kazlauskas A. A novel strategy to develop therapeutic approaches to prevent proliferative vitreoretinopathy. Am J Pathol 2011; 179:2931-40.}

In the course of investigating the pathogenesis of PVR, we learned that three different classes of growth factors engage PDGFRa, and that there was a hierarchy amongst them. Growth factors outside of the PDGF family (non-PDGFs, such as EGF, bFGF, IGF-1, etc.) indirectly activated monomeric PDGFRa. The direct (PDGF-mediated) and indirect modes of activating PDGFRa were incompatible. In the presence of both PDGFs and non-PDGFs, the direct mode dominated because PDGF assembled PDGFRa into dimers, which were poorly activated by the indirect mode. While the direct mode of activation predominated when non-PDGFs and PDGF were present, the addition of VEGF-A switched the mode of activation to indirect. This is because VEGF-A, which has a very similar crystal structure to PDGF-B, compete with PDGF for binding to PDGFRa and thereby sustains a population of monomeric PDGFRas in the face of PDGF. The relevance of this newly appreciated, indirect mode of activating PDGFRa relates to its ability to reduce p53 and thereby promote survival of cells. In so doing, indirectly activated PDGFRa can either promote pathology (in the case of PVR), or physiology (by enhancing survival of healthy tissue during periods of hypoxia)

_{Lei H, Kazlauskas A. Growth factors outside of the PDGF family employ ROS/SFKs to activate PDGF receptor alpha and thereby promote proliferation and survival of cells. J Biol Chem 2009; 284:6329-36.
Pennock S, Kazlauskas A. Vascular endothelial growth factor A competitively inhibits platelet-derived growth factor (PDGF)-dependent activation of PDGF receptor and subsequent signaling events and cellular responses. Mol Cell Biol 2012; 32:1955-66.
Pennock S, Haddock LJ, Mukai S, Kazlauskas A. Vascular Endothelial Growth Factor Acts Primarily via Platelet-Derived Growth Factor Receptor α to Promote Proliferative Vitreoretinopathy. Am J Pathol. 2014; 184: 3052-68.
Pennock S, Kim, L, Kazlauskas A. VEGF-A acts via PDGFRα to promote viability of cells enduring hypoxia. Mol Cell Biol. 2016 Aug 26;36(18):2314-27.}