Alejandro P. Adam, PhD

Associate Professor

Molecular and Cellular Physiology

Areas of Study

Disproportionate Inflammatory Responses

Education

University of Buenos Aires2007PhD
University of Buenos Aires1999BS/MS

Research

Our mission is to find novel therapeutics to treat tissue damage and organ dysfunction caused by disproportionate inflammatory responses. We aim to achieve that by identifying the most critical mechanisms of the pathophysiology that lead to chronic and excessive inflammation.

Find more info on Dr. Adam's website

When inflammation goes awry

A cytokine-driven acute increase in vascular permeability is an essential feature of the inflammatory response leading to tissue repair and pathogen clearance. However, sustained vascular leakage due to prolonged or exacerbated inflammation leads to organ damage, lasting sequelae and increased mortality. Multiple edemagenic factors are known to participate in the initial changes in endothelial permeability via well-defined mechanisms. The mechanism mediating the acute (minutes to hours) responses to proinflammatory cytokine signaling are well-described: phosphorylation of tyrosines in adherens junction proteins and an increase in actin-myosin contractility are two main mechanisms leading to a quick loss of endothelial intercellular adhesion and thus an increase in vascular permeability. Despite its importance in severe and chronic inflammation, little is known about the changes in the endothelium leading to a sustained (lasting hours to days) vascular barrier breakdown. Understanding these long-term mechanisms may prove crucial to allow us to directly target vascular leakage and minimize tissue damage, thus reducing the rates of mortality and chronic sequelae of excessive edema.

Endothelial signaling leading to edema – a role for STAT3

Multicellular organisms depend on strong, stable intercellular adhesion structures to maintain a cohesive state, but also on mechanisms to allow the separation of cells when required. Cells are held together by distinct cellular junctions, consisting of protein complexes that confer them with particular characteristics, such as protection against mechanical stress, barrier function, and cell-cell communication. These are the tight junction, the adherens junction (AJ), and the desmosome.

Our published and unpublished data led us to propose that the extent and intensity of proinflammatory signaling in the endothelium is subject to control by multiple canonical and non-canonical tyrosine and serine/threonine kinases, leading to drastic differences in the overall barrier function loss. It is well accepted that phosphorylation of tyrosines in adherens junction proteins and an increase in actin-myosin contractility are two main mechanisms leading to a quick loss of endothelial intercellular adhesion and thus an increase in vascular permeability. In addition, mechanisms such as NF-κB-dependent transcriptional regulation are known to contribute to the loss of barrier function hours after treatment with the proinflammatory cytokine TNF-α. The relative importance of each pathway (cell contraction, transcriptional regulation and adherens junction phosphorylation) is still a matter of debate and the level of crosstalk between these signaling mechanisms is poorly understood.

Despite the well-accepted role of IL-6 in inflammatory diseases, including vascular barrier breakdown, little is known about the signaling pathways required for IL-6-induced barrier loss, beyond the IL-6-induced activation of JAK kinase activity. IL-6 initiates signaling by binding to a heterodimeric receptor complex involving the gp80 (IL-6Rα) and gp130 subunits. Binding of the IL-6•IL-6Rα complex to gp130 allows gp130 to activate the JAK family of kinases (comprising JAK1, 2, 3 and TYK2) resulting in the stimulation of a number of downstream pathways. STAT3 phosphorylation at Y705 directly by JAK drives STAT3 dimerization, nuclear localization and binding to responsive promoter elements (23). In addition, JAK activity also promotes the recruitment of SHP2 and activation of PI3K/Akt and Raf/MEK/Erk pathways. In vivo, IL-6 and JAK signaling are critical factors in the development of edema following LPS or VEGF treatment as well as in models of heart ischemia/reperfusion, hemorrhagic shock, and focal cerebral ischemia. In vitro, IL-6-induced increases in permeability in the endothelium require JAK as demonstrated using inhibitors of JAK activity. We recently showed that IL-6 promotes an increase in endothelial monolayer permeability that lasts over 24 h and demonstrate that activation of Src and MEK/Erk pathways are required only for short-term increases in permeability, being dispensable after 2 h. Instead, JAK-mediated STAT3 phosphorylation at Y705 and de novo synthesis of RNA and proteins mediate the sustained increase in permeability.

Our current work is focused on determining how STAT3 can promote the sustained response and which downstream genes mediate the loss of endothelial barrier function. We identified several potential candidates in vitro and we are now testing whether these proteins mediate the drastic inflammatory response in two murine models of sepsis: systemic endotoxin and cecal ligation and puncture.

Endothelial signaling leading to edema – a role for STAT3

Innate immunity in rosacea

Rosacea is an incurable and highly prevalent disease that results in redness of the facial skin, discomfort, pain, damage to the eye, and vision loss. It affects over 16 million Americans. Although primarily considered a cutaneous condition, it may involve the eyes in up to 72% of patients. While patients of any age, race, and gender can be affected by rosacea, it primarily affects adults between 30 and 60 years of age, who are fair skinned and of European descent. The National Rosacea Society Expert Committee has recently moved away from the morphological characterization of this disease as four distinct subtypes, including erythematotelangiectatic rosacea, papulopustular rosacea, phymatous rosacea and ocular rosacea4, and updated the classification system to be based on phenotypes. Despite its common occurrence and potentially devastating effects, there is no successful medication to completely treat it, and the current therapies are ineffective and inadequate. When affecting the periorbital skin and eyelids, this disorder results in ocular surface manifestations, such as lid margin telangiectases, corneal infiltrates and conjunctival injection. Additionally this disorder represents a significant cause of social embarrassment and is an impediment to work for patients that suffer from it.

Recently, several studies have advanced our understanding of the pathophysiology leading to rosacea in general, and ocular rosacea in particular. We and others showed that an increased involvement of the innate immunity is associated with this disease. Using eyelid biopsies obtained from elective blepharoplasties from subjects with or without rosacea, we recently demonstrated a drastic increase in the activation of Erk and p38 MAPKs in the keratinocytes of these patients, suggesting a novel potential avenue to treat these patients. We are actively pursuing the development of this therapeutic strategy.

Facial edema and swelling is a common feature of rosacea, but the mechanisms leading to this vascular leak are not known. We hypothesized that this sustained loss may be also regulated by cytokine-induced changes in the endothelial transcriptional program. We are testing this hypothesis using a combination of in vitro and clinical research.

Comparison photo of control cells vs Rosacea cells with a chart below demonstrating the positive cells/field between the two

Edema in uveitis

Uveitis is a leading cause of blindness, accounting for approximately 10% of all cases of loss of sight. Uveitis is classified as anterior uveitis if it affects the iris or ciliary body, intermediate uveitis if presenting inflammation of the vitreous, or posterior uveitis when affecting the retina or choroid. Panuveitis refers to manifestations affecting most of the uvea. While rare in otherwise healthy individuals, uveitis can occur secondary to multiple causes, ranging from an infectious agent to autoimmune diseases. However, up to 50% of the cases remain idiopathic4. According to a study assessing insurance claims from over 4 million Americans, non-infectious uveitis accounts for over 90% of the cases. The retinal inflammation can cause macular edema, and in some cases choroid neovascularization, greatly impairing vision. A steroid treatment may cause immediate anti-inflammatory relief, but sustained use in chronic manifestations leads to the development of cataracts and glaucoma, thus limiting the therapeutic usefulness of these drugs. Anti-tumor necrosis factor (TNF-α) biologics, commonly used as treatment for multiple forms of arthritis for over a decade, showed efficacy to treat non-infectious uveitis and received FDA approval for its use in adults with non-infectious intermediate, posterior and panuveitis in 2016. Other FDA-approved biologics show also promising therapeutic potential for uveitis. Interleukin-6 (IL-6) is dramatically increased in the sera, aqueous and vitreous of uveitis patients. Further, some case studies and small clinical trials suggest that the use of IL-6 antagonists can alleviate the symptoms and improve vision in patients. These findings could be reproduced in animal models, where intravitreal injection of an anti-IL-6 attenuated experimental autoimmune uveitis. Studies performed in mice suggest that these biologics act at least in part by blocking Th17-mediated retinal inflammation. Consistently, loss of STAT3 in CD4+ T Cells prevented the development of experimental autoimmune uveitis in mice. Besides this insight, little is known about the mechanism of disease and the involvement of IL-6-induced pathways in disease progression. Moreover, any potential role for IL-6 signaling in the development of uveitic macular edema is currently unknown. Our current research is using transgenic mice to assess how the IL-6/STAT3 signaling axis regulates the development of macular edema during uveitis.

The endothelial adherens junction structure

Endothelial cells regulate the passage of fluids, macromolecules, and cells from the blood stream to the surrounding tissues. Endothelial permeability and transendothelial migration (TEM) are critical during acute and chronic inflammation, shock, atherosclerosis, acute lung distress syndrome, and primary tumor growth and metastasis. The EC is not only centrally involved in this process, but also readily exposed to circulating drugs and humanized antibodies, thus making it a perfect target for novel drug development. Hence, it is essential to understand its mechanisms of regulation in order to design better therapeutic strategies. Endothelial intercellular adhesion, and thus permeability, is tightly regulated by a number of vasoactive factors and is critical to determine the onset and extent of an inflammatory response. The endothelial AJ is comprised of the classical cadherin VE-cadherin and the catenins p120, β-catenin and plakoglobin. ECs contain AJ complexes along the cell-cell border in close association with a cortical actin ring, supporting strong cell-cell adhesion and resistance to the shear stress induced by the blood flow. Endothelial AJ can form very distinct structures within the cells, and their relative composition and function is currently unknown. Essentially, while we know the molecular partners of stable adherens junctions, little is known about the dynamic formation of these structures and why endothelial cells may show different AJ structures. The classical structure, termed here “linear” junction, is observed along the lines of cell-cell interactions in close association with the cortical actin ring. Linear junctions may occur together with “reticular” junctions, which are observed at regions of cell-cell overlap. These junctions are named after the distinct network of cadherins and catenins observed throughout the overlapping region. Linear AJ colocalize with tight junction markers, while reticular AJ do not. Further, linear junctions are less mobile than reticular junctions, suggesting that linear AJ may be more mature, stronger adhesion sites. Determining quantitatively the molecular composition of each structure is critical to be able to understand how endothelial cells interact and what is the role for each adherens junction structure. The lab is using several super-resolution strategies, including Airyscan and STED, and live tracking (mEOS2) in combination with AJ component mutants to better understand how the AJ forms and disassembles. The role for actin remodeling and focal adhesion components is also being studied.