PROJECT DESCRIPTION

Cardiovascular Diseases (CVDs) are the leading cause of morbidity and mortality worldwide. According to data released by the World Health Organization (WHO), 17.9 million people die from cardiovascular diseases each year, meaning that one in every three deaths is attributable to CVD. Additionally, data from the World Heart Federation (WHF) indicate that the global number of CVD patients has exceeded 500 million[1].

As reported in the China Health Statistics Yearbook 2022 (Fig.1), CVD ranks first in the composition of causes of death among both urban and rural residents in China. In 2021, CVD accounted for 48.98% and 47.35% of deaths in rural and urban areas, respectively, with the CVD mortality rate in rural areas consistently surpassing that of urban areas since 2009. Specifically, in 2021, the CVD mortality rate was 364.16 per 100,000 in rural areas and 305.39 per 100,000 in urban areas. Mortality rates from both heart disease and cerebrovascular disease were also higher in rural regions compared to urban areas.

Fig.1 Trends in CVD Mortality Rates: Urban vs. Rural Areas in China (2009-2021)

Despite significant advances in diagnostic technologies and drug development for cardiovascular diseases, clinical practice still faces two major challenges: low early diagnosis rates and a lack of precise early-warning tools.

Nitric oxide (NO) is a key molecule that maintains cardiovascular homeostasis and has gradually become a crucial breakthrough in the early detection of diseases. As a gaseous signaling molecule synthesized by vascular endothelial cells, NO is produced through the catalysis of L-arginine by nitric oxide synthase (NOS). Despite its extremely short half-life, NO plays multiple essential roles in the cardiovascular system: it mediates vasodilation by activating soluble guanylate cyclase (sGC) in smooth muscle cells, increasing cGMP and reducing calcium ion concentration; it significantly lowers the risk of thrombosis by inhibiting the glycoprotein IIb/IIIa receptor on platelets; additionally, NO suppresses the NF-κB pathway to reduce the release of inflammatory factors and scavenges superoxide anions, exhibiting both anti-inflammatory and antioxidant effects[2]. Meanwhile, NO maintains vascular structural stability by regulating the proliferation and migration of vascular smooth muscle. Its metabolic dysfunction is closely associated with the development and progression of diseases such as atherosclerosis, hypertension, and heart failure, making it a "barometer" of cardiovascular health.

A decline in NO bioavailability is an early event in cardiovascular pathology. Oxidative stress can cause endothelial nitric oxide synthase (eNOS) uncoupling, leading to reduced NO production, while excessive reactive oxygen species (ROS) generation further accelerates its degradation, forming a vicious cycle that ultimately results in endothelial dysfunction and atherosclerotic plaque formation. Studies have shown that decreased NO levels enhance macrophage infiltration and induce the release of matrix metalloproteinases (MMPs), disrupting plaque stability[3]. In eNOS gene knockout hypertensive models, mice exhibited a significant increase in systolic blood pressure, confirming that impairment of the NO/cGMP pathway is a key mechanism underlying elevated vascular resistance[4]. NO also influences myocardial energy metabolism by regulating the mitochondrial respiratory chain, and its deficiency exacerbates cardiomyocyte apoptosis and fibrosis during heart failure progression[5]. These findings consistently indicate that dynamic changes in NO not only precede structural lesions but also integrate multiple pathological pathways such as oxidative stress, inflammation, and metabolism, highlighting its potential as an ideal early warning biomarker.

However, the clinical translation of NO has long been hindered by limitations in detection technologies. Existing methods such as electron paramagnetic resonance, chemiluminescence, colorimetry, and electrochemical sensors often face significant challenges in sensitivity, specificity, real-time capability, or biocompatibility. These techniques are generally costly, require complex operational procedures, and struggle to achieve spatiotemporally resolved dynamic analysis in living cells or in vivo environments. In recent years, advances in genetically encoded fluorescent sensor technology have offered new strategies to address this issue. These probes can specifically bind to target molecules and generate fluorescent signals, enabling precise spatiotemporal monitoring of small biological molecules within subcellular structures[6]. Although several fluorescent NO probes have been reported, they still suffer from notable limitations in dynamic response range and specificity. Thus, there remains an urgent need to develop high-performance genetically encoded NO sensors.

This project aims to develop a novel genetically encoded fluorescent probe for nitric oxide (NO). The probe is constructed based on a fusion protein comprising the regulatory domain of the bacterial transcription factor NorR and a fluorescent protein. It will be systematically optimized through random mutation and directed evolution strategies to achieve a highly dependent and specific fluorescent response to NO concentrations.

Furthermore, by incorporating subcellular targeting signal sequences, the probe will be directed to specific regions such as the cytoplasm and mitochondria, enabling high-resolution in situ imaging of NO in living cells. This tool is expected to provide critical technical support for the early detection of subclinical cardiovascular pathologies, the evaluation of therapeutic efficacy, and the development of targeted drugs.

Our research plan involves systematic development and optimization of the genetically encoded NO fluorescent probe. We will start with the initial design and construction of the fusion protein, followed by directed evolution to enhance sensitivity and specificity. We will also develop subcellular targeting variants for different cellular compartments to enable comprehensive NO monitoring within the cell.

The probe will be validated using various cellular and animal models of cardiovascular diseases, with a focus on demonstrating its utility for early disease detection and therapeutic monitoring. Additionally, we will work on integrating the probe with portable detection devices to facilitate clinical applications.