Biology subjects

Hanjaya-Putra, D.

Publications and source records attributed to Hanjaya-Putra, D..

6 recordsLinked to original sources

bioRxiv2026

Harnessing NCX-IP3R-dependent Calcium Oscillations to Regulate Angiogenic Signaling in Endothelial Cells

The blood vasculature has a high capacity for structural regeneration, driven by the blood endothelial cells (BECs) that comprise it. This regenerative process, which involves BEC migration and proliferation to form these complex tissues, is linked to low frequency (< 0.1 Hz) calcium spiking that precedes these activities. However, we need new approaches to stimulating angiogenic responses in tissue engineering applications. By conducting experiments that manipulate local ionic concentrations and developing a simple, yet powerful, computational analysis, we demonstrate that sodium-calcium cross-talk is a crucial component that regulates the calcium signaling and downstream angiogenic responses. Activation and deactivation of the inositol triphosphate 3 receptors (IP3Rs) on the endoplasmic reticulum (ER) and the switch between forward and reverse modes of the sodium-calcium exchanger (NCX) are proposed to be the key mechanisms underlying calcium oscillations when cells are exposed to temporary cationic depletion. The spiking is suggested to be a release of intracellular calcium mediated by IP3R activity, and transport in or out of the cell is driven by NCX for the calcium oscillatory signaling pattern. The NCX and IP3R both contribute to manage intracellular calcium and ionic concentration as initially there is a long ER deactivation period while intracellular sodium slowly increases until a sudden onset of calcium is released by the ER. Other calcium and sodium ion channels can change this resonant coupling of ER and NCX to alter the inter-spike duration. Synchronization of the spiking intervals between cells is triggered by stimulating with vascular endothelial growth factor (VEGF), which induces a propagating wave of intracellular calcium across the 2D tissue culture, prior to coordinated cell migration and proliferation towards the VEGF source. This wave, which can be artificially induced and studied using electrical stimulation, suggests that the underlying sodium-calcium crosstalk mechanism introduces intracellular calcium polarization, whose orientation is transferred across cells through spike synchronization. Thus, control of calcium signaling dynamics through regulation of ionic depletion can serve as useful method for generating angiogenic responses in engineered tissue constructs.

cell biology↗

bioRxiv2025

Glutamine Availability Impacts Lymphatic Endothelial Cell Glycolysis and Lymphangiogenesis in Hypoxic Environments

Dysfunctional lymphangiogenesis is a component of several diseases, including secondary lymphedema, allergic asthma, and solid malignancies. These vessels are ineffective at draining interstitial fluid, resulting in secondary complications such as edema, increased inflammation, slowed wound healing, and, for cancer patients, increased risk of metastasis. Hypoxic microenvironments drive dysfunctional vessel growth by upregulating vascular endothelial growth factor receptors, migratory signaling pathways, and glycolytic metabolism. Despite the potential benefits of normalizing lymphatic vasculature, most treatments for vascular normalization focus on blood vasculature, ignoring the unique properties of lymphatic endothelial cells (LECs). Moreover, previous targets for vascular normalization center around vascular endothelial growth factors, which risk adverse side effects. To address these issues, we approached lymphatic normalization from a metabolic perspective. In this study, we investigated the impact of glutamine availability on factors critical to lymphangiogenesis, including glycolysis, cell proliferation, and migration. We found that increasing the concentration of glutamine in media results in increased lactate production and the expression of glycolytic genes HK2, GLUT1, and GLUT3 under hypoxia. The presence of glutamine also encouraged LEC proliferation, while blocking glutamine transport reduced lactate production, HK2 expression, and slowed collective LEC migration. In a 2D vessel formation assay, we found that glutamine increased vessel formation in normoxic conditions but lowered vessel connectivity in hypoxic conditions, reflecting the dysfunction seen in hypoxic diseases. However, attenuating glycolysis by blocking glutamine transport caused LECs to form longer, interconnected vascular networks. This study reveals that glutamine availability can modulate LEC glycolysis, and therefore lymphangiogenesis, in a hypoxia-dependent manner.

cell biology↗

bioRxiv2025

Metabolically driven differentiation of human pluripotent stem cells to generate robust and functional lymphatic endothelial cells

The lymphatic system plays various crucial but underappreciated roles in fluid transport and immune response in numerous organs and tissue types. Consequently, generation of lymphatic vessels has been postulated as an innovative therapeutic strategy for various diseases. However, there is a lack of efficient and reliable method to differentiate human pluripotent stem cells into lymphatic endothelial cells (LEC) for lymphatic regeneration. Current differentiation methods suffer from poor yield and low lymphatic marker expression, while also having limited clinical applicability due to its reliance on either the embryoid body intermediates or xenogenic supporting cells. Given that LECs exclusively rely on anaerobic and fatty acid metabolism due to the hypoxic environment of the lymph, here we report that the unique lymphatic-specific metabolic pathways can be exploited to promote lymphatic identity in differentiated LECs (dLECs). We show that dLECs express elevated levels of lymphatic markers compared to native endothelial cells, which is up to 15 times higher than the current leading standard of dLECs. Moreover, dLECs can form lymphatic vascular networks in both 2D and 3D, as well as secrete important lymphangiocrine for organ maturation. Upon implantation into double-ligation tail lymphedema and mammary fat pad models, dLECs were able to integrate with the host lymphatic vessels, restore fluid flow, and reduce swelling. Collectively, we show that metabolite supplementation can drive stem cell differentiation into dLECs, which can be incorporated into new alternative methods for personalized therapies and disease modeling, as well as provide a direct therapeutic option for lymphedema and lymphatic disorders. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/686405v2_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1c4caa5org.highwire.dtl.DTLVardef@d42b08org.highwire.dtl.DTLVardef@1554ffcorg.highwire.dtl.DTLVardef@1f64ae6_HPS_FORMAT_FIGEXP M_FIG C_FIG A xeno-free and stepwise differentiation protocol with VEGF-C and sodium acetate induces lymphatic identity in iPSC-derived endothelial cells. Differentiated LECs (dLECs) express key lymphatic markers as verified using bulk RNA-sequencing, qPCR, FACS, and immunostaining. These dLECs secrete important cytokines for lympangiocrine signaling, able to form robust 2D and 3D lymphatic networks, as well as demonstrate in vivo functionality and host integration in murine models.

bioengineering↗

bioRxiv2024

Intracellular pH dynamics respond to microenvironment stiffening and mediate vasculogenic mimicry through β-catenin

Dysregulated intracellular pH (pHi) dynamics and an altered tumor microenvironment have emerged as drivers of cancer cell phenotypes. However, the molecular integration between the physical properties of the microenvironment and dynamic intracellular signaling responses remains unclear. Here, we identify a mechanistic link between ECM stiffness and pHi dynamics in driving vasculogenic mimicry (VM), an aggressive cancer phenotype associated with poor prognosis. We performed single-cell imaging of pHi in lung and breast metastatic cell lines cultured on tunable-stiffness hydrogel systems. We used two tunable-stiffness hydrogel systems to independently model stiffness induced by increased protein secretion (Matrigel) and increased protein crosslinking (Hyaluronic acid gels). We show that increased ECM stiffness lowers single-cell pHi in both lung and breast metastatic cell lines. We also observed that stiff ECM promotes a distinct morphological phenotype called vasculogenic mimicry (VM). Importantly, we show that low pHi is a necessary mediator of VM, as raising pHi on stiff ECM reduces VM phenotypes. We also find that lowering pHi on soft ECM was sufficient to induce VM in the absence of extracellular stiffening. We characterized {beta}-catenin as a pH-dependent molecular mediator of VM, where stiffness-driven increases in {beta}-catenin abundance can be overridden by high pHi, which destabilizes {beta}-catenin to reduce VM on stiff ECM. In contrast, the transcription factor FOXC2 is activated by ECM stiffness but is insensitive to pHi, and its activity alone is insufficient to maintain VM at high pHi when {beta}-catenin is lost. We uncover a novel mechanotransduction axis in which ECM stiffness regulates intracellular pH to drive {beta}-catenin-induced VM. We also show pHi dynamics can override mechanosensitive cell responses to the extracellular microenvironment. Thus, our work positions pHi as an integrator of mechanotransduction in cancer, suggesting a new framework for therapeutically targeting pHi in cancer and perhaps in other diseases driven by ECM remodeling.

cell biology↗

bioRxiv2024

Optimal Performance Objectives in the Highly Conserved Bone Morphogenetic Protein Signaling Pathway

Throughout development, complex networks of cell signaling pathways drive cellular decision-making across different tissues and contexts. The transforming growth factor {beta} (TGF-{beta}) pathways, including the BMP/Smad pathway, play crucial roles in these cellular responses. However, as the Smad pathway is used reiteratively throughout the life cycle of all animals, its systems-level behavior varies from one context to another, despite the pathway connectivity remaining nearly constant. For instance, some cellular systems require a rapid response, while others require high noise filtering. In this paper, we examine how the BMP- Smad pathway balances trade-offs among three such systems-level behaviors, or "Performance Objectives (POs)": response speed, noise amplification, and the sensitivity of pathway output to receptor input. Using a Smad pathway model fit to human cell data, we show that varying non-conserved parameters (NCPs) such as protein concentrations, the Smad pathway can be tuned to emphasize any of the three POs and that the concentration of nuclear phosphatase has the greatest effect on tuning the POs. However, due to competition among the POs, the pathway cannot simultaneously optimize all three, but at best must balance trade-offs among the POs. We applied the multi-objective optimization concept of the Pareto Front, a widely used concept in economics to identify optimal trade-offs among various requirements. We show that the BMP pathway efficiently balances competing POs across species and is largely Pareto optimal. Our findings reveal that varying the concentration of NCPs allows the Smad signaling pathway to generate a diverse range of POs. This insight identifies how signaling pathways can be optimally tuned for each context.

systems biology↗

bioRxiv2023

The Effects of Preeclamptic Milieu on Cord Blood Derived Endothelial Colony-Forming Cells

Preeclampsia is one of the leading causes of infant and maternal mortality worldwide. Many infants born from preeclamptic pregnancies are born prematurely with higher risk of developing cardiovascular later in their life. A key mechanism by which these complications occur is through stress-induced dysfunction of endothelial progenitor cells (EPCs), including endothelial colony-forming cells (ECFCs). To gain insight into this, cord blood derived ECFCs isolated from preeclamptic pregnancies (PRECs) were analyzed and compared to their healthy counterparts. While PRECs preserve key endothelial markers, they upregulate several markers associated with oxidative stress and inflammatory response. Compared to ECFCs, PRECs also exhibit lower migratory behaviors and impaired angiogenic potential. Interestingly, treatment of neuropilin-1 can improve tube formation in vitro. Collectively, this study reports that preeclamptic milieu influence phenotypes and functionality of PRECs, which can be rejuvenated using exogenous molecules. Promising results from this study warrant future investigations on the prospect of the rejuvenated PRECs to improve lung function of infants born from preeclamptic pregnancies.

bioengineering↗