Kidney cancer is the 7th and 10th most common cancer in men and women in the United States respectively. Renal cell carcinoma (RCC), a cancer of the proximal convoluted tubule, accounts for >90% of these kidney cancer incidences. Most cancers, including RCC, stimulate angiogenesis – the sprouting of existing blood vessels. By upregulating the expression of angiogenic factors, such as vascular endothelial growth factor (VEGF), tumors promote vascular sprouting within their microenvironment. This increased vasculature allows for increased oxygen transport as well as an opportunity to metastasize. This tumor-induced angiogenesis is of particular interest to researchers hoping to continue to develop alternative cancer therapeutics to inhibit angiogenesis, starving the tumor of oxygen, or normalize the vasculature, making drug delivery more effective. Common cancer models like 2D cell cultures and animal xenografts have been shown to be poor predictors of drug efficacy in patients. A lack of cellular complexity in 2D cultures along with genetic differences in animal xenografts make them insufficient models of the human pathophysiology seen in vivo. To address this problem, we are developing a 3D, vascularized, flow directed, microphysiological device deemed a “kidney-tumor-on-a-chip”. To engineer a perfusable microvessel, a 120μm collagen channel was lined with human umbilical vein endothelial cells (HUVECs). Primary RCC spheroids that mimic the 3D structure of solid tumors were then introduced into the surrounding collagen, recapitulating the tumor-vessel microenvironment. Initial results have shown no endothelial sprouting in non-RCC control devices, compared to significant endothelial sprouting in devices with RCC spheroids. These results suggest RCC cells may contribute to angiogenic sprouting in our microphysiological device, simulating what is observed in vivo. The ability of this kidney-tumor-on-a-chip to mimic the tumor-vessel microenvironment demonstrates its potential to be a more effective cancer model for drug testing.

Quantum dots (QDs), fluorescent semiconductor nanocrystals, can be used as a biomarker and diagnostic tool because of their unique optical and electrical properties, including high luminescence, resistance to photobleaching, and broad excitation and emission spectrums. But in order to use QDs as biomedical imaging devices, particularly in sensitive organs such as the developing brain, toxicity must be adequately characterized. Little is known about the toxic effect of QDs on the brain, especially on the neonatal brain. QDs consist of a metal core, typically cadmium selenide, and a shell to protect the core. Cadmium is known to be highly toxic to many of the body’s systems, including the central nervous system. However, expanding the shell and attaching functional biomolecules prevents cadmium release and changes the characteristics of the particles. In our study, several surface ligands were tested at variable concentrations and periods of exposure to measure the toxic potential of QDs on an ex vivo organotypic brain slice model. Three methods of characterization were used: a lactate dehydrogenase (LDH) assay, a propidium iodide stain, and a fluoro-jade C stain. The cellular stains were evaluated using confocal microscopy. Results showed that toxicity is a function of QD exposure time, surface chemistry, and concentration. Several QD conjugations displayed minimal or no toxic effect after 24 h of exposure. The results of this study will be useful in identifying a QD conjugate as nontoxic in a biological setting. QDs can then be tailored to site and cell-specific uptake in the brain, thereby improving the selectivity of current imaging techniques and providing a powerful diagnostic with regards to diseased cell fate.

Quantum dot (QD) semiconductor nanocrystals offer significant advantages over conventional fluorescent markers due to broad excitation spectrum, narrow emission spectrum, and very high photo stability, which enables long term visualization. Because of this, there is great interest in using QDs as biomarkers, where cellular uptake of QDs play an important role. In this study, we aim to characterize QD uptake in neuronal and glial populations in the developing brain as a function of QD surface functionality. Previous research has shown that the behavior of QDs in biological systems can be dictated by surface functionality; however, this has not been systematically studied, particularly in the developing brain. Here, we used cadmium-selenide (CdSe) QDs with either cadmium-sulfide (CdS) or zinc-sulfide (ZnS) shell. These QDs are coated with a layer of surface ligands to protect the core-shell and minimize their hydrophobicity. We set out to examine five different surface coatings, including: mercaptoundecanoic acid (MUA), mercaptopropionic acid (MPA), polyethylene glycol-amine (PEG-NH2), polyethylene glycol-methoxy (mPEG) and carboxylic acid (COOH). We incubated organotypic neonatal rat brain slices in 0.1 and 0.01 µM of QDs over a 24 h period. Using immunohistochemistry and co-localization analysis, we observed QDs with PEG-NH2 functionality localize in neurons, whereas QDs with MUA or MPA surface functionality remain in the brain extracellular space. High resolution confocal imaging is used to visually assess the stained brain slices, and the degree of colocalization with cell stains can be quantified using ImageJ. Furthermore, we investigated the region-dependent of QD-PEG-NH2 localization in neurons. This provides insight into the mechanism of uptake for future studies, which include using QDs as biomarkers of inflammatory processes, mediated by glia, in the developing brain.

In Plasmodium falciparum malaria, infected erythrocytes (IEs) adhere to vascular endothelium, causing occlusion, coagulopathy, organ dysfunction and inflammation. While IE cytoadhesion occurs in many organs, cerebral malaria in particular is associated with high IE cytoadhesion and life-threatening complications. However, IE cytoadhesion in cerebral malaria remains incompletely understood. Research into human cerebral malaria pathogenesis has been limited by the inaccessibility of the human brain, and existing experimental platforms fail to recapitulate human vascular conditions and disease pathophysiology. In vitro microfluidic models lack appropriate geometry and materials to mimic in vivo flow conditions and matrix, while animal models do not adequately replicate human disease pathology due to use of non-human malaria parasites and non-human tissues. To address these limitations, we propose a 3D in vitro brain microvascular model to study P. falciparum cerebral malaria pathogenesis. Microvessels are created by molding collagen I with micropatterned stamps and incorporating brain endothelium and supporting cells into the collagen channel. Micropatterned stamps allow specification of complex physiological vessel network structure, and when these networks are designed using computational flow simulation, they allow specification of physiological flow properties in the fabricated microvessel. Collagen comprises the vascular extracellular matrix in vivo and regulates cell morphology, lumen formation and vessel stability, so its use as a biomaterial scaffold increases physiological relevance of the in vitro model. By constructing a physiologically relevant brain microvascular bed, we will characterize the molecular and biophysical mechanisms of cytoadhesion and the resulting changes in local endothelial function. The completion of this project will improve understanding of IE cytoadhesion, thereby improving understanding of P. falciparum pathophysiology to guide the development of treatments targeting disease mechanisms.

There are many age-related cardiac diseases that relate to impairment of muscle function, including heart failure and arrhythmia. Previous studies show that increased mitochondrial function results in the enhancement of cardiac and skeletal muscle function as mice age. In these studies, the SS-31 tetrapeptide was used, an agent that serves as an antioxidant by binding to cardiolipin in the inner mitochondrial membrane, enhancing electron transport and increasing ATP generation, and protecting against damage from Reactive Oxygen Species (ROS) by reducing their generation. However prior literature has suggested that ultrastructural morphology does not change in relation to age. The goal of this study is to quantitatively assess interfibrillar mitochondrial damage caused by these ROS', and to see if there is structural improvement after treatment with the SS-31 peptide. Eight 24 month-old mice were split into two treatment groups, four treated with SS-31 for 8 weeks and four untreated. Four 8 month-old untreated mice were used as controls for youthful, “ideal” mitochondrial structure. The mice were euthanized, and cardiac tissue was harvested, sectioned, and examined by transmission electron microscopy (TEM). Quantitative analysis using NIH ImageJ software allowed the comparison of the SS-31 treated and untreated old hearts to young hearts. This revealed stark differences when comparing the young and old ultrastructure. Untreated old mice exhibited higher amounts of vacuolation, greater lack of mitochondrial axial organization, and lower density of mitochondrial cristae than the younger untreated mice. Older mice treated with SS-31 appear to exhibit improvement in ultrastructural definition compared to untreated older mice, but additional quantitation is necessary to validate this observation. In further testing, we are examining the persistence of the SS-31 tetrapeptide's positive effects after its administration period ends, and how ultrastructure changes during this refractory period.

Endothelial cells (ECs) are cells that line our blood vessel walls. They differ throughout the circulatory system. For example, high pressure arteries typically have tighter endothelial junctions/gaps than the leaky, low pressure veins. ECs are known to sense fluid shear stress and hydrostatic pressure. These cues play a large part in embryonic development of the vasculature and induce short term responses in adults, such as dilation and constriction of arterioles, but it is unknown if adult ECs adapt to them in the long term. This matters for many medical procedures, such as venous graft bypass surgery, where the saphenous vein is implanted to bypass a blocked artery. These grafts are often lifesaving successes, but they sometimes re-occlude. Venous and arterial endothelium are very distinct, and it is important to know if the venous ECs in the graft can adapt to their new arterial environment. If not, that could explain the pathological re-occlusion. Here, we measure adaptation by changes in gene expression from a venous to an arterial phenotype. We designed a novel in vitro pump system to expose a flow chamber with endothelial cells to controlled flow and pressure. HUVECs (human umbilical vein endothelial cells) were then cultured and seeded into the chamber. Finally, a quantitative real time polymerase chain reaction (qRT-PCR) will measure gene expression at various time points in the venous (20 cmH₂O, low flow) and arterial (60 cmH₂O, high flow) cases. We analyze the markers EphrinB4 (EphB4) (higher in veins), EphB2 (arteries), Cx40 (veins) and endomucin (arteries). We present preliminary gene expression data from the 24-hour time point. We hypothesized that HUVECs will respond to arterial conditions with increased arterial gene expression, while retaining their venous state in venous conditions. Supporting or falsifying this will illuminate endothelial cell response to changing conditions in the body.

Vascular remodeling, the modification of preexisting vasculature, often occurs during events such as cardiovascular disease. Little is known about the perivascular factors controlling vascular remodeling. A small-diameter, single-channel arteriole model was developed to study vascular remodeling under various perivascular conditions, such as those of disease models or under the addition of drugs. The housing device consists of polydimethylsiloxane (PDMS), a non-toxic silicone-based polymer, held by a plexiglass casing. Type I collagen gel embedded with human coronary artery smooth muscle cells (HCASMCs) is cast within the housing, and the lumen is formed using a needle-based subtractive molding method with a 180um acupuncture needle. The original diameter of the lumen is retained while human umbilical vein endothelial cells (HUVECs) are seeded into the lumen by hand-perfusion. Migration and realignment of both the HCASMCs and the HUVECs is observed after applying physiological flow rates of growth media for 3 days in co-culture. Additionally, endothelial barrier function and smooth muscle contractile function are maintained. The functionality of the model is confirmed through observed calcium wave propagation and epinephrine treatments, where dilation of the arteriole occurs upon perfusion of the hormone. Current studies include observing the effects of combined pressure and flow on perivascular remodeling and function with a focus on modeling atherosclerosis and arterial thrombosis. This study will demonstrate the role of pressure in controlling the vascular remodeling process, specifically the migration and alignment of HCASMCs and HUVECs. It will also provide insight into vascular remodeling during coronary artery disease.

Recent research has been dedicated to investigating ways to promote angiogenesis using microphysiological systems that more accurately represent native organs. The Zheng lab has recently created a kidney microvascular model on a chip that utilizes primary isolated endothelial cells. However, this model requires the implementation of a biologically representative matrix and the incorporation of perivascular and parenchyma cells to better mimic the native microenvironment. We hypothesize that the incorporation of a biomimetic matrix will confer quiescence in the endothelium of the microvascular model and that the integration of perivascular cells within the matrix will promote improved vessel maturation and the establishment of a basal lamina. We have synthesized a biomimetic material from decellularized human kidney tissue that has a complex modulus of 930 Pa, making it stiff enough to use in semi-3D cell culture. Furthermore, use of this material has led to decreased endothelial migration and fewer alpha smooth muscle actin positive perivascular cells, indicating representative in vivo behavior of both cell types. In the microvascular model, we expect the integration of perivascular cells to the matrix will lead to increased endothelial quiescence. Results in collagen I matrices have shown irregular perivascular homing to and coverage of the endothelium, denoting increased but not complete vascular maturation. We expect the utilization of this technique in the biomimetic matrix to show increased perivascular coverage of the endothelium and the formation of a more complete basal lamina. If proven to be biologically accurate, implications of an in vitro kidney microphysiological model include use in therapeutic tissue regeneration studies as well as the ability to replace less accurate animal models during drug testing.