Biology subjects

Koch, K. S.

Publications and source records attributed to Koch, K. S..

4 recordsLinked to original sources

bioRxiv2026

Tunable Laminar Perfusion Coordinates Endothelial and Perivascular Remodeling in Angiogenic Vasculature-on-Chip

Perfusable vascular microphysiological systems are increasingly used to model angiogenesis, tissue crosstalk, and disease. However, many platforms still rely on oscillatory, discontinuous, or poorly controlled perfusion regimes, limiting the study of sustained flow-dependent vascular remodeling. Here, we establish a tunable, unidirectional laminar flow workflow for long-term perfusion of angiogenic vasculature-on-chip cultures and use it to investigate endothelial, perivascular, and immune cell responses to sustained flow. Using an AIM Biotech microfluidic platform containing 14-day-old human umbilical vein endothelial cell-derived angiogenic sprouts and pericytes, continuous perfusion enabled intraluminal transport of 1 m tracer beads through vessels, demonstrating stable flow across the vascular bed. Sustained laminar flow induced endothelial remodeling at both the mother vessel and sprout levels, with cellular alignment evident in both compartments. Quantitative analysis of the mother vessel further revealed Golgi polarization against the direction of flow. Sustained perfusion also increased pericyte recruitment to angiogenic sprouts and reduced endothelial proliferation within the mother vessel, consistent with flow-driven vascular maturation and quiescence. Live-cell imaging further captured directional endothelial migration against the flow, lumen remodeling, and dynamic pericyte behavior under continuous perfusion. In immune-cell assays performed under continuous-flow conditions, interactions with untreated endothelium were limited, whereas inflammatory activation increased immune-cell adhesion and crawling. These observations suggest that sustained flow supports a quiescent endothelial phenotype and demonstrate the suitability of the platform for studying inflammatory activation and immune-vascular communication under controlled hemodynamic conditions. Beyond its biological relevance, the workflow provides practical advantages for live-cell imaging, low medium consumption, and downstream perturbation studies. Moreover, the modular design of the platform makes it well suited for vascular-organ crosstalk applications. Collectively, these results establish laminar flow angiogenic vasculature-on-chip as an experimentally tractable model for studying vascular mechanobiology, vascular maturation, and dynamic cell interactions under defined hemodynamic conditions.

bioengineering↗

bioRxiv2024

N-Acetyl-2-Aminofluorene (AAF): In vivo temporal expression patterns of growth cycle-dependent macromolecular binding constants KD are revealed by primary cultures of premalignant hepatocytes derived from a multi-cyclic hepatocarcinogenic feeding regimen

Biochemical investigations of the hepatoprocarcinogen N-acetyl-2-aminofluorene (AAF) have shown that normal adult rat hepatocytes in primary culture express two sets of pharmacokinetic constants - designated Systems I and II, and Sites I and II - associated respectively with the metabolism (System I [high-affinity Km[APPARENT] and low-velocity VMAX[APPARENT]] and System II [low-affinity Km APPARENT] and high-velocity VMAX[APPARENT]]), and the macromolecular binding (Site I [high-affinity KD[APPARENT] and low capacity BMAX[APPARENT]] and Site II ([low-affinity KD[APPARENT] and high-capacity BMAX[APPARENT]]) of AAF. Additional findings - that genomically saturating levels of AAF-DNA adducts form far below reported extracellular AAF concentrations required to block replicative and repair DNA synthesis; and, that biphasic Site I and Site II BMAX[APPARENT] and KD[APPARENT] expression curves varied inversely with respect to time and magnitude during hepatocyte growth - led us to wonder how macromolecular binding constants are expressed during chemical hepatocarcinogenesis. These questions were addressed by Scatchard analysis measurements through five consecutive AAF feeding cycles. Notably, cultured premalignant hepatocytes displayed reduced and elevated BMAX[APPARENT] and KD[APPARENT] levels, respectively, akin to the Site I and Site II expression curves observed during hepatocyte growth transitions in vitro. In contrast, prominent hepatocellular functions such as N-OH-AAF production, DNA replication, cell aggregation and resistance to AAF toxicity displayed different temporal trajectories. Impact StatementStriking similarities are observed between both Site I and Site II BMAX and KD expression curves during in vitro and in vivo premalignant growth transitions. These new findings fit earlier ones that hepatocytes growing during carcinogen exposure manifest fewer intranuclear AAF-DNA adducts. How this phenomenon leads to malignancy remains unclear.

pharmacology and toxicology↗

bioRxiv2017

N-acetyl-2-aminofluorene (AAF) processing in adult rat hepatocytes in primary culture occurs by high-affinity low-velocity and low-affinity high-velocity AAF metabolite-forming systems

N-acetyl-2-aminofluorene (AAF) is a procarcinogen used widely in physiological investigations of chemical hepatocarcinogenesis. Its metabolic pathways have been described extensively, yet little is known about its biochemical processing, growth cycle expression and pharmacological properties inside living hepatocytes - the principal cellular targets of this hepatocarcinogen. In this report, primary monolayer adult rat hepatocyte cultures and high specific-activity [ring G-3H]-N-acetyl-2-aminofluorene were used to extend previous observations of metabolic activation of AAF by highly differentiated, proliferation-competent hepatocytes in long-term cultures. AAF metabolism proceeded by zero-order kinetics. Hepatocytes processed significant amounts of procarcinogen ({approx}12 g AAF/106 cells/day). Five ring-hydroxylated and one deacylated species of AAF were secreted into the culture media. Extracellular metabolite levels varied during the growth cycle (days 0-13), but their rank quantitative order was time invariant: 5-OH-AAF > 7-OH-AAF > 3-OH-AAF > N-OH-AAF > aminofluorene (AF) > 1-OH-AAF. Lineweaver-Burk analyses revealed two principal classes of metabolism: System I (high-affinity and low-velocity), Km[APPARENT] = 1.64 x 10-7 M and VMAX[APPARENT] = 0.1 nmols/106 cells/day; and, System II (low-affinity and high-velocity), Km[APPARENT] = 3.25 x 10-5 M and VMAX[APPARENT] = 1000 nmols/106 cells/day. A third system of metabolism of AAF to AF, with Km[APPARENT] and VMAX[APPARENT] constants of 9.6 x 10-5 M and 4.7 nmols/106 cells/day, was also observed. Evidence provided in this report and its companion paper suggests selective roles and intracellular locations for System I- and System II-mediated AAF metabolite formation during hepatocarcinogenesis, although some of the molecules and mechanisms responsible for multi-system processing remain to be fully defined.

cancer biology↗

bioRxiv2017

High-affinity low-capacity and low-affinity high-capacity N-acetyl-2-aminofluorene (AAF) macromolecular binding sites are revealed during the growth cycle of adult rat hepatocytes in primary culture

Long-term cultures of primary adult rat hepatocytes were used to study the effects of N-acetyl-2-aminofluorene (AAF) on hepatocyte proliferation during the growth cycle; on the initiation of hepatocyte DNA synthesis in quiescent cultures; and, on hepatocyte DNA replication following the initiation of DNA synthesis. Scatchard analyses were used to identify the pharmacologic properties of radiolabeled AAF metabolite binding to hepatocyte macromolecules. Two classes of growth cycle-dependent AAF metabolite binding sites - a high-affinity low-capacity site (designated Site I) and a low-affinity high-capacity site (designated Site II) - associated with two spatially distinct classes of macromolecular targets, were revealed. Based upon radiolabeled AAF metabolite binding to purified hepatocyte genomic DNA or to DNA, RNA, proteins and lipids from isolated nuclei, Site IDAY 4 targets (KD[APPARENT] {approx} 2-4 x 10-6 M and BMAX[APPARENT] {approx} 6 pmols/106 cells/24 h) were consistent with genomic DNA; and with AAF metabolized by a nuclear cytochrome P450. Based upon radiolabeled AAF binding to total cellular lysates, Site IIDAY 4 targets (KD[APPARENT] {approx} 1.5 x 10-3 M and BMAX[APPARENT] {approx} 350 pmols/106 cells/24 h) were consistent with cytoplasmic proteins; and with AAF metabolized by cytoplasmic cytochrome P450s. DNA synthesis was not inhibited by concentrations of AAF that saturated DNA binding in the neighborhood of the Site I KD. Instead, hepatocyte DNA synthesis inhibition required higher concentrations of AAF approaching the Site II KD. These observations raise the possibility that carcinogenic DNA adducts derived from AAF metabolites form below concentrations of AAF that inhibit replicative and repair DNA synthesis.

cancer biology↗