Vascular endothelial growth factor, or VEGF-A, has a complicated reputation in lung biology. The factor was originally discovered as vascular permeability factor, a molecule secreted by tumor cells that made blood vessels leak fluid at an alarming rate. In the lung, that leakiness is precisely the problem in acute lung injury: when the endothelial lining of pulmonary microvessels becomes permeable, protein-rich fluid floods the airspaces, oxygen exchange collapses, and patients may require mechanical ventilation to survive. Earlier clinical studies had found elevated VEGF in the airspaces of patients with respiratory distress syndrome, and researchers have long debated whether the growth factor is a culprit driving vascular leak or, paradoxically, a repair signal that helps the damaged endothelium recover. The new findings suggest that part of the confusion may have arisen because investigators were measuring a family of molecules with opposite effects as if it were a single entity.

The key to that complexity lies in alternative splicing, the process by which a single gene yields multiple protein versions. In the final stretch of the VEGF-A gene, two different sets of terminal exons can be stitched onto the coding sequence. One choice produces the classical pro-angiogenic isoforms, exemplified by VEGF165a, which bind the receptor VEGFR2 on endothelial cells and trigger the signaling cascades that make vessels grow and leak. The alternative choice produces the so-called b-variants, including VEGF165b, which differ in just six amino acids at the carboxyl terminus. That subtle change, first characterized by researchers studying kidney and cancer biology in the early 2000s, is enough to render the variant weakly angiogenic and poorly permeability-inducing, while still allowing it to occupy the same receptor. The b-isoforms therefore behave as natural antagonists of their pro-angiogenic siblings, competing for receptor binding without delivering the same intracellular message.

The Guangzhou-led team began by asking whether this splice balance is disturbed in human lung injury. Analyzing blood samples from patients, they found that the circulating ratio of VEGF165b to total VEGF-A was significantly reduced in acute lung injury, and that the lower the proportion of the protective variant, the more severe the disease. That inverse correlation, reported alongside ethical approval from the First Affiliated Hospital of Guangzhou Medical University and Zhujiang Hospital of Southern Medical University, provided the first hint that a relative deficiency of VEGF165b might accompany, and possibly contribute to, the vascular catastrophe of respiratory distress syndrome. Correlation alone cannot establish causation, so the investigators turned to experimental models in which the relationship could be tested directly.

In mice, the researchers induced acute lung injury with lipopolysaccharide, the inflammatory toxin derived from bacterial cell walls that mimics Gram-negative sepsis, one of the most common triggers of the human syndrome. When the animals received exogenous VEGF165b, the consequences of the insult were markedly blunted: pulmonary edema diminished, inflammatory infiltration of the lung tissue decreased, and overall measures of injury improved. The converse experiment proved equally telling. When the team neutralized endogenous VEGF165b with antibodies, leaving the pro-inflammatory isoforms to act unopposed, lung damage became worse. Together, these gain-of-function and loss-of-function results established the splice variant as a genuine endogenous protector rather than a passive bystander, and they identified the VEGF165b-to-total-VEGF ratio as a candidate biomarker for tracking disease severity.

The mechanistic core of the study explains how a single splice decision can translate into barrier protection at the molecular level. Using human microvascular endothelial cells, human pulmonary artery endothelial cells, and primary pulmonary microvascular endothelial cells, the researchers showed that VEGF165b acts as a competitive antagonist at VEGFR2, selectively blocking the phosphorylation of the receptor at tyrosine 1175, a docking site whose activation is known to be essential for the permeability response. With that phosphorylation event suppressed, the downstream PI3K/AKT/eNOS pathway, which normally drives nitric oxide production and vessel relaxation during vascular leak, was quieted. The team traced the consequences further downstream to the Src kinase and the adherens junction protein VE-cadherin, the molecular glue that holds endothelial cells together. When Src phosphorylates VE-cadherin, the junctions loosen and fluid escapes; by preventing the upstream signal, VEGF165b reduced VE-cadherin phosphorylation and thereby kept the endothelial barrier intact.

Intriguingly, the protective variant does not stop at junctional reinforcement. Through RNA sequencing and network analysis of the signaling hubs affected by VEGF165b, the investigators found that the variant also suppresses the chemokine CCL2, also known as monocyte chemoattractant protein 1. CCL2 is a potent recruiter of circulating monocytes, and its reduction translated into a second, distinct anti-inflammatory mechanism: fewer monocytes adhered to the endothelial surface, limiting the wave of inflammatory cells that normally amplifies tissue damage in acute lung injury. The authors describe this as a dual mode of action, in which VEGF165b simultaneously strengthens the physical wall of the microvasculature and dampens the cellular inflammatory assault against it. Such a combination is rare among candidate therapies, which typically address either leakiness or inflammation but rarely both.

The findings also reframe a long-running therapeutic debate. Several anti-VEGF strategies, including the monoclonal antibody bevacizumab, have been explored for severe COVID-19 and other causes of pulmonary edema on the logic that blocking VEGF should reduce vascular leak. But blanket VEGF blockade carries well-documented risks, including hypertension, proteinuria, and thromboembolism, precisely because the growth factor performs essential maintenance roles in healthy vessels. The splice-variant view offers a more surgical alternative: rather than eliminating VEGF signaling altogether, one could in principle restore the balance between pro-permeability and anti-permeability isoforms, either by administering recombinant VEGF165b, which has already been tested as an anti-cancer agent in animal models, or by shifting the splicing machinery toward the protective variant. The study’s demonstration that neutralizing VEGF165b worsens injury adds a note of caution for any therapy that removes this natural brake indiscriminately.

As with any preclinical advance, the road from mouse models and cell cultures to the intensive care unit is long. The patient data establish an association between a low VEGF165b fraction and disease severity, but clinical trials will be needed to determine whether replenishing the variant improves outcomes, at what dose, and at what stage of illness. Questions also remain about how the splicing balance is regulated in the acutely inflamed lung, and whether splicing factors such as the SRSF family, which other studies have implicated in acute lung injury, could serve as drug targets. Nevertheless, the conceptual payoff is substantial. The work reveals that vascular homeostasis in the lung depends on the critical equilibrium of VEGF splice variants, positions VEGF165b as both a biomarker and a potential therapeutic agent for acute lung injury and respiratory distress syndrome, and offers a new strategy for treating vascular barrier dysfunction in critical lung disease, one of the most stubborn unsolved problems in modern critical care medicine.

Subject of Research: The role of the VEGF-A splice variant VEGF165b in protecting against acute lung injury by antagonizing VEGF165a/VEGFR2 signaling

Article Title: VEGF165b attenuates LPS-induced acute lung injury via antagonizing the VEGF165a/VEGFR2 signaling pathway

Article References: Deng, J., Wang, X., Wu, Z., Bai, Q., Luo, Q., Zhou, L., Yao, Y., Wang, H., Zhong, K., Liu, H., Chen, X., & Wang, T. (2026). VEGF165b attenuates LPS-induced acute lung injury via antagonizing the VEGF165a/VEGFR2 signaling pathway. Angiogenesis, 29(4), Article 75. https://doi.org/10.1007/s10456-026-10090-4

Image Credits: AI Generated

DOI: 10.1007/s10456-026-10090-4

Keywords: acute lung injury, ARDS, VEGF165b, VEGFR2, vascular permeability, endothelial barrier, alternative splicing, VE-cadherin, CCL2, lipopolysaccharide, angiogenesis, pulmonary edema

The 1,3,4-oxadiazole is a compact five-membered ring containing two nitrogen atoms and one oxygen atom, and it has long been a favourite building block in medicinal chemistry because it appears in compounds with antibacterial, antifungal, anti-inflammatory and anticancer activity. The researchers, led by Rekha Singh and Jagadish Singh of Guru Ghasidas Vishwavidyalaya in Bilaspur, together with Balaji Wamanrao Matore and colleagues, drew on two decades of their own prior work with this scaffold. Their design strategy placed a phenyl ring directly on the oxadiazole core, a motif also found in the approved drug zibotentan, and attached a second chlorinated phenyl ring through an imine, or CH=N, linker at the five-position of the ring.

Synthesis proceeded in three straightforward steps. Substituted aldehydes were first condensed with semicarbazide hydrochloride in ethanol to form Schiff base intermediates, which were then cyclised with iodine and potassium carbonate in 1,4-dioxane at 80 degrees Celsius to build the oxadiazole ring. A final condensation with different chloro-substituted aldehydes delivered the nine target compounds, R1 through R9, in yields above 90 percent. Every product was confirmed by FT-IR, proton NMR and mass spectrometry, with the diagnostic imine proton appearing between 8.73 and 8.99 parts per million in the NMR spectra, and melting points recorded for each purified solid.

Before any wet-lab testing, the team ran a battery of computational experiments. Density functional theory calculations using the DMol3 code suggested that all nine compounds are more chemically reactive than the kinase inhibitor sorafenib, with R3, R4 and R6 showing the smallest HOMO-LUMO gaps at 0.086 Hartree, a measure that correlates with a molecule’s willingness to engage in electronic interactions. Target prediction servers, including PASS and SwissTargetPrediction, then pointed the researchers toward kinase receptors, a family of enzymes heavily implicated in breast cancer biology through receptors such as VEGFR2, EGFR, PDGFR and HER2.

VEGFR2, the vascular endothelial growth factor receptor 2, emerged as the priority target because of its central role in angiogenesis, the process by which tumours recruit new blood vessels to fuel their growth and spread. Blocking VEGFR2 is a clinically validated strategy in breast cancer management, and the researchers selected the crystal structure 4ASD from the Protein Data Bank for docking studies. After validating their protocol by re-docking the co-crystallised ligand and achieving root-mean-square deviations well below the accepted two angstrom threshold, they docked all nine compounds into the ATP binding pocket using three independent algorithms: CDOCKER, LibDock and AutoDock Vina.

The docking results told a remarkably consistent story. Compound R3 achieved the highest CDOCKER interaction energy at 41.54 and the best LibDock score at 130.38, and it formed fifteen separate contacts with the protein, including hydrogen bonds to GLU885, ASP1046 and LYS868, three residues known to stabilise ATP-competitive inhibitors at the hinge region of the kinase. Twelve additional hydrophobic contacts with residues such as LEU840, VAL848, PHE918 and CYS1045 locked the molecule deep in the pocket. Fourteen of these interactions were shared with the co-crystallised reference ligand, meaning R3 essentially mimics the binding mode of a proven VEGFR2 inhibitor. Binding free energy calculations confirmed the picture, with R3 showing the most favourable stability of the series at minus 84.42 kilocalories per mole, compared with minus 142.01 for sorafenib.

Structure-activity relationship analysis revealed the decisive role of chlorine positioning. Derivatives bearing meta and para chloro substituents on their phenyl rings, compounds R1 through R4, consistently outperformed those with ortho substitution, compounds R5 through R9. The explanation is steric: an ortho chlorine crowds the space near the imine linker and prevents the molecule from adopting the flat, extended conformation needed to slot into the VEGFR2 pocket. The ortho-substituted compounds formed almost no favourable interactions in docking, showed weaker binding free energies, and translated this deficit directly into higher IC50 values in the cell assays. It is a textbook example of how a single atom, moved a few angstroms, can flip a molecule from active to inert.

The computational predictions were then put to the test in the laboratory. Using the MTT assay against MCF-7 breast cancer cells at concentrations ranging from 5 to 80 micromolar, all nine compounds showed moderate to potent, concentration-dependent cytotoxicity, with IC50 values spanning 8.068 to 17.343 micromolar. Three compounds, R1, R3 and R4, fell below the 10 micromolar threshold, and R3 led the series at 8.068 micromolar, close to the 4.051 micromolar recorded for doxorubicin under the same conditions. Microscopy of treated cultures showed reduced cell density and altered morphology relative to untreated controls, consistent with the viability data.

Pharmacokinetic modelling added a further layer of optimism. SwissADME predictions indicated high gastrointestinal absorption and good oral bioavailability for the whole series, with moderate lipophilicity, LogP values of 3.3 to 3.5, and a low topological polar surface area of 51.28 square angstroms, a balance well suited to membrane permeability. The ProTox-II server classified all compounds in toxicity class 4 with an LD50 of 375 milligrams per kilogram and predicted them to be non-carcinogenic, non-mutagenic and non-immunotoxic, although moderate hepatotoxicity was flagged for the series, mirroring the profile of sorafenib itself. The authors note two caveats for future optimisation: moderate aqueous solubility may complicate formulation, and the predicted ability to cross the blood-brain barrier raises the possibility of off-target central nervous system exposure that must be managed in an anticancer context.

The study’s authors are careful to frame the work as an early-stage screening effort. MCF-7 served as the initial model, and broader evaluation across additional cancer cell lines and normal cell models will be needed to establish selectivity and safety, particularly for R3, which the team identifies as the lead candidate for further development. Even so, the internal consistency of the data is compelling: the same substitution pattern that produced the best docking scores also produced the strongest binding free energies and the lowest IC50 values, and the key residues engaged by R3 match those exploited by clinically approved VEGFR2 inhibitors such as sorafenib. For a scaffold as chemically accessible as the 1,3,4-oxadiazole, synthesised in three steps from cheap starting materials with yields above 90 percent, that combination of potency, predictability and synthetic simplicity makes R3 a lead worth watching as VEGFR2-targeted breast cancer drug discovery moves forward.

Subject of Research: Design and evaluation of 1,3,4-oxadiazole derivatives as VEGFR2-targeted anticancer agents against breast cancer

Article Title: Design, synthesis, SAR, in silico and in vitro evaluation of 1, 3, 4-oxadiazole derivatives against breast cancer

Article References: Singh, R., Matore, B. W., Murmu, A., Lakra, N., Banjare, P., Namdeo, K. P., Roy, P. P., & Singh, J. (2026). Design, synthesis, SAR, in silico and in vitro evaluation of 1, 3, 4-oxadiazole derivatives against breast cancer. Discover Chemistry, 3(1), Article 539. https://doi.org/10.1007/s44371-026-00898-w

Image Credits: AI Generated

DOI: 10.1007/s44371-026-00898-w

Keywords: breast cancer, 1,3,4-oxadiazole, VEGFR2, MCF-7, molecular docking, structure-activity relationship, ADMET, drug discovery, angiogenesis, kinase inhibitors, MTT assay, medicinal chemistry

The study, published in the journal Angiogenesis, focuses on a long-standing puzzle in cancer biology: how tumors recruit and remodel blood vessels to sustain their growth. Tumors cannot enlarge beyond a few millimeters without recruiting new vasculature, a process known as tumor angiogenesis. The vessels that form inside tumors are typically abnormal — leaky, tortuously branched, and poorly covered by supportive pericyte cells — which creates hypoxic, acidic conditions that paradoxically stimulate even more angiogenic signalling, most notably through vascular endothelial growth factor, or VEGF, and its receptor VEGFR2. The new work reveals that Epac1 sits at a central junction of this process, coordinating both the chemical VEGF signals and the mechanical forces that shape tumor vessel formation.

Epac1, encoded by the gene RAPGEF3, is a guanine nucleotide exchange factor activated by the intracellular messenger cyclic AMP. It exists in two isoforms, but only Epac1 is expressed in endothelial cells, where it participates in regulating junctional dynamics and cell polarity. To determine whether Epac1 matters in tumor angiogenesis specifically, the team first analyzed publicly available single-cell RNA sequencing data from human primary melanomas and healthy human skin. They found that RAPGEF3 expression was markedly enriched in the endothelial cells of melanoma tissue compared with the endothelium of healthy skin, and that the gene was predominantly active in vascular rather than lymphatic endothelial cells. This human evidence suggested that the protein might be a genuine feature of the tumor vasculature rather than an artifact of experimental models.

The researchers then turned to mouse models. In mice lacking Epac1 throughout the body, subcutaneously implanted B16F10 melanoma cells still formed palpable tumors, but growth slowed dramatically after day fourteen — precisely the stage at which vascularization becomes the limiting factor for tumor expansion. By day eighteen, tumors in the knockout animals were roughly half the size of those in normal mice, and staining for the endothelial marker CD31 revealed a significantly smaller vascular area within them. Because global deletion could not distinguish effects in blood vessel cells from effects in other stromal cells, the team generated a second mouse line in which Epac1 could be deleted selectively in endothelial cells using a tamoxifen-inducible Cre system. The result was the same: reduced tumor volume and reduced vascularization, confirming that the endothelial supply of Epac1 itself drives melanoma growth.

To understand the molecular mechanism, the researchers isolated tumor endothelial cells from the melanomas and performed whole-transcriptome sequencing. In the Epac1-deficient vessels, gene ontology analysis showed broad downregulation of angiogenesis-associated pathways, including sprouting angiogenesis, endothelial cell migration, and proliferation. Key pro-angiogenic genes such as Kdr (which encodes VEGFR2), Angpt1, Hif1a, Dll1, and Dll4 were all reduced. Strikingly, the analysis also revealed suppressed expression of canonical target genes of the transcriptional co-activators YAP and TAZ — including Ccn1, Ccn2, and Ankrd1 — pointing to a previously unknown connection between Epac1 and the Hippo signalling pathway in tumor vessels.

YAP and TAZ are mechanosensitive transcriptional regulators that act as major effectors of the Hippo pathway. Their activity depends less on how much protein is present and more on where the protein resides: when they translocate into the nucleus, they partner with TEAD-family transcription factors to switch on genes that promote cell proliferation, migration, and survival. In the Epac1-deficient endothelial cells, the researchers found that total YAP and TAZ protein levels were unchanged, but nuclear localization was significantly reduced, and TEAD-dependent reporter activity was markedly diminished. A parallel experiment using a selective pharmacological inhibitor of Epac1’s enzymatic activity in human umbilical vein endothelial cells produced the same suppression of YAP/TAZ target gene expression, confirming that the catalytic function of the protein is required to maintain this transcriptional program.

The team then traced how Epac1 links VEGF signalling to YAP/TAZ activation. In control endothelial cells, VEGF stimulation robustly induced expression of Vegfr2 and Ccn2, activated the small GTPase RhoA, and promoted phosphorylation of myosin light chain 2, a key downstream target of the RhoA effector kinase ROCK. In Epac1-deficient cells, all of these responses were blunted or abolished. Pharmacological inhibition of RhoA with C3 transferase reduced TEAD-driven transcription and suppressed VEGF-induced Ccn2 expression, while VEGF-driven nuclear translocation of YAP and TAZ in human endothelial cells was fully prevented by RhoA inhibition. Together, these experiments establish a signalling chain running from Epac1 through VEGFR2, RhoA, and the actin cytoskeleton to the nucleus, where YAP and TAZ execute their pro-angiogenic gene program.

Perhaps the most striking findings concerned mechanosensing — the ability of endothelial cells to respond to physical forces such as blood flow. Tumor vessels experience disturbed, oscillatory flow patterns that promote pro-angiogenic signalling and metastasis. Using an in vitro system that applies oscillatory shear stress to endothelial monolayers, the researchers showed that control cells aligned themselves with the direction of flow, whereas Epac1-deficient cells completely failed to do so. Under the same conditions, oscillatory shear stress normally increases the interaction between VE-cadherin, the junctional adhesion protein, and VEGFR2 — a partnership thought to form part of the endothelial mechanosensory complex. Proximity ligation assays revealed that this interaction was abolished in Epac1-deficient cells, as was shear-induced phosphorylation of VE-cadherin at tyrosine 658, an essential modification for mechanosensing, and the shear-induced nuclear accumulation of YAP and TAZ. When the researchers reintroduced Epac1 into the knockout cells using lentiviral vectors, VEGFR2 and Ccn2 expression, VEGF responses, and flow-induced alignment were all rescued, confirming the specificity of the effect.

The study also carries an important nuance about Epac1’s role in vascular biology. Previous work by the same group and others has shown that Epac1 is dispensable during physiological angiogenesis — the retinal vascular area of Epac1-deficient mice at postnatal day five is comparable to that of wild-type animals — yet pathological neovascularization in oxygen-induced retinopathy is significantly reduced without the protein. The new melanoma data fit this pattern: endogenous endothelial Epac1 appears to be largely irrelevant for normal vascular maintenance but becomes functionally indispensable in pathological settings characterized by excessive VEGF signalling and altered mechanical cues. This context-dependence is encouraging from a therapeutic standpoint, because it suggests that drugs targeting Epac1 might impair tumor vessel growth while sparing the normal vasculature, potentially reducing the side effects that complicate current anti-angiogenic therapies.

The authors caution that their study used melanoma as the tumor model, and that the prominent upregulation of Epac1 in tumor endothelium and its consequences for tumor growth may not generalize to all cancer types. Nevertheless, the identification of Epac1 as a central modulator that integrates VEGF signalling, RhoA-dependent cytoskeletal dynamics, YAP/TAZ transcription, and flow mechanosensing provides the most complete mechanistic picture to date of how a single endothelial protein can orchestrate pathological tumor vascularization. With Epac inhibitors already under investigation for other cancers, the prospect of starving melanomas by dismantling the molecular machinery of their blood vessels has moved a tangible step closer to the clinic.

Subject of Research: The role of endothelial Epac1 in regulating VEGFR2 and YAP/TAZ signalling during melanoma angiogenesis

Article Title: Endothelial Epac1 facilitates YAP/TAZ controlled melanoma growth and angiogenesis

Article References: Wibowo, Y. C., Ma, N., Ren, Y., Cordero, J., Gahn, J., Chen, Z., Levay, M., Ola, R., Feng, Y., Dobreva, G., Langer, H., Wieland, T., Vettel, C., & Jansen, S. (2026). Endothelial Epac1 facilitates YAP/TAZ controlled melanoma growth and angiogenesis. Angiogenesis, 29(4), Article 72. https://doi.org/10.1007/s10456-026-10080-6

Image Credits: AI Generated

DOI: 10.1007/s10456-026-10080-6

Keywords: Epac1, melanoma, angiogenesis, tumor endothelial cells, VEGFR2, YAP/TAZ, Hippo signalling, RhoA, mechanotransduction, VE-cadherin, VEGF, skin cancer