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protease activity is regulated by specialized inhibitors, with serpins representing the largest and most diverse
family, consisting of over 1,000 identified members across species. Serpins mimic potential substrates and
form irreversible, stable complexes with proteases through a suicide mechanism, leading to loss of catalytic
function. Other endogenous inhibitors, such as secretory leukocyte protease inhibitor (SLPI), elafin, and α-
macroglobulins, provide an additional layer of regulation . The multilevel regulation of serine protease
[45]
activity ensures a balance between proteases and their inhibitory mechanisms, which is necessary for the
maintenance of homeostatic equilibrium and tissue integrity. In the next part, we will discuss how
inadequate control of serine proteases can be a central component of the pathogenesis of vascular
remodeling in PAH.

NEUTROPHIL SERINE PROTEASES IN PAH
Neutrophil serine proteases (NSPs) are a class of granule-associated enzymes essential for intracellular
pathogen killing. However, upon cell activation NSPs can also be released into extracellular space where
they degrade ECM components and activate inflammatory mediators. The role of NSPs in the inflammatory
response is supported by the observations made in transgenic mice. Animals that lack either NSPs or the
enzyme that regulates their maturation, i.e., cathepsin C, show an impaired inflammatory response [48,49] . The
most abundant serine proteases in neutrophil granules are neutrophil elastase (NE), cathepsin G (CatG),
and proteinase 3 (PR3). Although NSPs are produced primarily by neutrophils, it is important to note that
other immune cells, including B cells, monocytes, macrophages, and mast cells, can also produce these
[45]
enzymes .
Neutrophil elastase (NE)
Neutrophils are a dominant source of NE. However, expression of this serine protease can also be detected
in structural cells, e.g smooth muscle cells and endothelial cells . The increased neutrophil-to-leukocyte
[50]
[51]
[41]
ratio is commonly found in PAH patients . The observed difference in neutrophils is not only quantitative
but also qualitative. When compared to the healthy controls, neutrophils isolated from the blood of PAH
[52]
patients respond to stimulation with enhanced release of NE . A recent observational study in the PAH
cohort confirmed that increased serum levels of NE, with a concomitant deficiency in NE inhibitor (elafin),
can be found across PAH subtypes . Furthermore, a high NE level has a prognostic value and is associated
[53]
[53]
with a worse clinical outcome, making circulating NE a promising PAH biomarker . NE is thought to
contribute to vessel remodeling in several ways. First, neointimal lesions show altered ECM composition
and fragmented elastic lamina [7,54] . Elastolytic activity within the vessel generates elastin peptides and
releases ECM-bound growth factors. These products can provide a chemotactic signal for the recruitment of
immune cells and stimulate the proliferation and migration of PASMCs [55,56] . NE can contribute to
remodeling not only by targeting elastin; it also cleaves many other ECM proteins, i.e., collagens,
fibronectin, and laminin , and activates different proteolytic enzymes, e.g., matrix metalloproteinases
[57]
[58]
(MMPs), which potentiate matrix degradation and remodeling . Furthermore, neutrophil proteases
propagate inflammatory signals via the modulation of the bioactivity of cytokines and chemokines . NSPs
[59]
are crucial components of the antimicrobial response. They are capable of effectively killing bacteria, but
this occurs typically in the micromolar concantration commonly found within phagolysosomes. However,
the concentration of proteases released during the degranulation of activated neutrophils is likely much
lower [9,60,61] . It is noteworthy that even at very low concentrations, neutrophil-derived proteases can activate
the IL-1 cytokine family, indicating that proteases released during degranulation have an important role in
[61]
regulating the inflammatory response rather than serving primarily as effectors in microbial killing .
Inflammatory tissue injury starts with the adhesion of neutrophils to vascular intima, and the association
between endothelial dysfunction, characterized by altered vasodilatory capacity, cell survival and growth
factor production, and PAH is firmly established . NE has been shown to damage the protective glycocalyx
[24]