Blood Pressure Drug Model L-NAME Directly Pushes Vascular Smooth Muscle Cells Toward Foam Cell State

A widely used laboratory compound that mimics hypertension-related blood vessel damage can drive vascular smooth muscle cells to accumulate fat and switch on atherosclerosis-linked molecular programs all by themselves, without any change in blood pressure or flow, according to a new in vitro study published in BMC Molecular and Cell Biology. The finding, reported by a team led by Gang Chen, suggests that the nitric oxide pathway disrupted in high blood pressure may also act directly on the muscle cells of the artery wall, potentially accelerating the earliest cellular steps of plaque formation.

The compound in question, L-NAME (N-nitro-L-arginine methyl ester), is a competitive inhibitor of nitric oxide synthase enzymes, the cellular machinery that produces nitric oxide. Nitric oxide is a gaseous signaling molecule central to vascular health: it promotes vasodilation, inhibits platelet aggregation, limits leukocyte adhesion, and restrains the growth of vascular smooth muscle cells. When nitric oxide bioavailability falls, vessels constrict, peripheral resistance rises, and hypertension follows. In animal research, chronic L-NAME administration also activates the renin-angiotensin system, raises angiotensin II signaling, promotes oxidative stress, and enhances vascular inflammation. Because of these cascading systemic effects, scientists have long debated whether L-NAME’s apparent pro-atherogenic influence in living animals is merely a byproduct of elevated blood pressure and endothelial dysfunction, or whether the compound can act on smooth muscle cells directly.

To isolate that question, the researchers turned to MOVAS cells, a mouse aortic vascular smooth muscle cell line obtained from a commercial supplier and maintained under standard culture conditions. Because these cultures contain no endothelial cells and experience no mechanical stress from blood flow, any response observed can be attributed to the compound’s direct action on the smooth muscle cells themselves. The authors caution, however, that MOVAS cells may express low or variable levels of nitric oxide synthase isoforms, which complicates interpretation of exactly which molecular target is being inhibited in this setting.

The experimental design was deliberately conservative. Before any mechanistic work, the team ran a cell viability assay using the CCK-8 reagent, testing L-NAME across a wide concentration range from 6.25 to 800 micromoles per liter and oxidized low-density lipoprotein, or ox-LDL, from 6.25 to 800 micrograms per milliliter over 24 hours. Ox-LDL served as the positive atherogenic control, a well-established inducer of vascular smooth muscle dysfunction that works through intracellular reactive oxygen species generation, NF-kappaB activation, inflammatory cytokine expression, lipid accumulation, and phenotype modulation. The viability screen showed no significant difference from untreated controls at L-NAME concentrations of 100 micromoles per liter or below, and at ox-LDL concentrations of 100 micrograms per milliliter or below. Cytotoxicity emerged at 200 micromoles per liter for L-NAME and 200 micrograms per milliliter for ox-LDL, with viability falling to roughly 50 percent at the highest doses. To ensure that any observed changes reflected specific cellular responses rather than nonspecific consequences of cell death, the team selected the highest concentrations preserving at least 90 percent viability: 50 micromoles per liter of L-NAME and 100 micrograms per milliliter of ox-LDL.

With those non-toxic working concentrations established, the first major test examined lipid accumulation using Oil Red O staining, a classic histochemical method in which neutral lipids appear as bright red round droplets under the microscope. After 24 hours of treatment, control cells showed minimal to no staining, retaining the clear cytoplasm and classic spindle shape characteristic of healthy vascular smooth muscle cells. In striking contrast, L-NAME-treated cells accumulated significant numbers of bright red, spherical lipid droplets filling both the perinuclear and peripheral cytoplasm of a large proportion of cells, a morphology consistent with transformation into foam cells. Foam cell formation, once attributed exclusively to macrophages, is now recognized as an early key event in atherogenesis when it occurs in vascular smooth muscle cells. The ox-LDL-treated cells showed qualitatively similar lipid loading, confirming that the model responds as expected to a known foam cell-inducing stimulus. The authors note that the extent of L-NAME-induced accumulation was higher than they had anticipated.

The second line of investigation probed whether L-NAME also shifted the cells’ molecular identity. In healthy arteries, vascular smooth muscle cells occupy a contractile, quiescent state defined by proteins such as alpha-smooth muscle actin, encoded by the ACTA2 gene, and smooth muscle myosin heavy chain 11, encoded by MYH11. Under atherogenic stimuli, these cells can transition to a synthetic phenotype marked by down-regulation of contractile proteins and up-regulation of synthetic markers such as secreted phosphoprotein 1, or SPP1. Synthetic-phenotype cells gain the capacity to proliferate, migrate, and produce extracellular matrix components and matrix metalloproteinases, and this phenotypic switch is considered an important early step in fibrous cap formation, plaque progression, luminal narrowing, and plaque instability.

Quantitative real-time polymerase chain reaction revealed exactly that pattern. L-NAME treatment reduced ACTA2 mRNA to approximately 40 percent of control levels and significantly lowered MYH11 mRNA, both at p less than 0.05. Meanwhile, SPP1 mRNA rose approximately 3.5-fold above control levels, also at p less than 0.05. Immunofluorescence staining for alpha-smooth muscle actin protein corroborated the transcript data: control cells displayed organized, filamentous, strong green staining consistent with a contractile phenotype, whereas L-NAME-treated cells showed markedly decreased fluorescence with a fragmented, disorganized residual signal. Quantitative analysis of mean optical density, measured across at least five randomly selected fields per coverslip, confirmed significantly lower alpha-SMA protein expression in the L-NAME group at p less than 0.001. Ox-LDL produced directionally similar changes across all of these markers.

The study also documented an up-regulation of matrix-remodeling genes. Matrix metalloproteinases MMP-2 and MMP-9 are zinc-dependent endopeptidases that degrade basement membrane and interstitial matrix components, including type IV collagen and gelatin, thereby facilitating smooth muscle cell migration and contributing to fibrous cap remodeling in atherosclerotic lesions. After 24 hours of L-NAME exposure, MMP-2 and MMP-9 mRNA expression rose significantly compared with controls, with MMP-9 increasing approximately four-fold from baseline. Ox-LDL likewise up-regulated both enzymes. All experiments drew on three independent biological replicates per condition, with one-way ANOVA followed by Tukey’s post-hoc test used for statistical comparisons.

The authors are careful to delineate what their data do and do not establish. Because the study did not measure nitric oxide synthase isoform expression, endothelial NOS activity, or intracellular nitric oxide production in the MOVAS cells, the findings cannot prove that eNOS inhibition alone mediates the observed effects. The team also did not assess reactive oxygen species generation, angiotensin II production, AT1 receptor activation, NF-kappaB, MAPK, or RhoA/ROCK signaling, and no rescue experiments with nitric oxide donors, reactive oxygen species scavengers, or AT1 receptor blockers such as losartan were performed. Functional assays were equally absent: no scratch migration, transwell migration, proliferation, gelatin zymography, or extracellular matrix degradation tests were run, so the results establish marker-based phenotypic modulation rather than full functional confirmation of a synthetic phenotype. Only a single 24-hour time point was examined, leaving open whether lipid accumulation precedes or follows the marker changes.

Further limitations temper the translational reach of the work. The MOVAS line is a clonal murine cell model that does not fully recapitulate the heterogeneity of primary mouse or human vascular smooth muscle cells, and validation in primary cells from both species will be needed. The authors frame their conclusions accordingly: L-NAME directly promotes lipid accumulation, contractile marker loss, synthetic phenotype marker induction, and increased matrix-remodeling gene expression in these cells under non-cytotoxic in vitro conditions, supporting the concept that disruption of the nitric oxide pathway may contribute to smooth muscle cell dysfunction independently of systemic hemodynamic effects. Future studies, they write, should determine whether the responses are mediated by eNOS-dependent nitric oxide depletion, other NOS isoforms, oxidative stress, angiotensin II signaling, or convergent downstream mechanisms. If confirmed, the convergence of L-NAME and ox-LDL on overlapping pro-atherogenic programs would suggest that distinct vascular risk stimuli, including the nitric oxide deficiency associated with hypertension, may funnel into shared cellular pathways that push artery-wall muscle cells toward the foam cell fate.

Subject of Research: Direct effects of the NOS inhibitor L-NAME on foam cell formation and phenotype marker expression in vascular smooth muscle cells in vitro

Article Title: L-NAME promotes foam cell formation and synthetic phenotype marker changes in vascular smooth muscle cells independent of hemodynamic effects

Article References: Du, H., Ling, J., Lu, Z., Liu, Y., Yan, J., Ma, J., Li, L., & Chen, G. (2026). L-NAME promotes foam cell formation and synthetic phenotype marker changes in vascular smooth muscle cells independent of hemodynamic effects. BMC Molecular and Cell Biology, 27(1), Article 38. https://doi.org/10.1186/s12860-026-00600-z

Image Credits: AI Generated

DOI: 10.1186/s12860-026-00600-z

Keywords: L-NAME, vascular smooth muscle cells, foam cells, atherosclerosis, nitric oxide, NOS inhibition, phenotype switching, MMP-2, MMP-9, ox-LDL, MOVAS cells, hypertension