# Hemolysis and Free Heme Signaling in Pulmonary Hypertension

> **NIH NIH R01** · INDIANA UNIVERSITY INDIANAPOLIS · 2024 · $742,328

## Abstract

Hemolysis is one of the critical pathogenic mechanisms of pulmonary hypertension (PH),
predisposing individuals with hemolytic conditions to PH. Notably, 10-30% of sickle cell disease patients
and up to 80% of thalassemia patients develop PH, starkly contrasting to a mere 0.001% prevalence
in the general population. Moreover, our recent findings indicate significant correlations between
elevated sub-clinical hemolysis in Group 1 PAH patients and PAH severity. However, the critical
mechanism of heme actions leading to PH progression still needs to be understood. By elucidating the
impact of heme on the pulmonary vasculature, we aim to shed light on the contribution of heme-
mediated mechanisms to the pathogenesis of Group 1 PAH.
 Our previous studies demonstrated that the free heme directly triggers intracellular signaling in
endothelial cells by activating the MKK3/p38 pathway. However, the precise mechanism by which free
heme activates this signaling axis remains to be determined. Our search for heme-sensor factors
identified a Heme-Activating Protein (HAP1) responsible for sensing the heme and initiating heme
target genes in Yeast. The homolog of HAP1 in humans is the INAVA, known to be responsible for
triggering an immune response in macrophages and disrupting the barrier in epithelial cells.
 However, there is a gap in knowledge on the molecular mechanism of INAVA action in
endothelial cells and its involvement in hemolytic complications and PH pathogenesis. Based on our
compelling preliminary data, we introduce INAVA as a novel intracellular heme-sensor protein activating
MKK3/p38-mediated signaling by engaging 14-3-3 kinase. Notably, both INAVA and 14-3-3 are
overexpressed in the lung tissues of PAH patients and have increased interaction, implying a disease-
related signaling role. We aim to examine the hypothesis that free heme, by binding to INAVA, induces
14-3-3 activation in the cytosol. This increases endothelial proliferation, barrier dysfunction, and
cytokines, leading to PH pathogenesis.
 Our research effort, focused on investigating endothelial cell heterogeneity using a single-cell
approach, allowed us to discern the distinct pulmonary endothelial cell populations, each possessing
unique gene signatures and functional arrays. We found five novel populations of general capillary ECs,
named gCapA-E, in addition to well-known pulmonary arterial, venous, lymphatic endothelium, and
aerocytes. Based on our preliminary data, we hypothesize that heme possesses a differential effect
on each of these endothelial cell populations, compromising the maintenance of adequate endothelial
barrier by gCapA, impairing angiogenic properties of gCapC, and altering proliferation of gCapB.

## Key facts

- **NIH application ID:** 10980934
- **Project number:** 2R01HL151447-06
- **Recipient organization:** INDIANA UNIVERSITY INDIANAPOLIS
- **Principal Investigator:** Ruslan Rafikov
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $742,328
- **Award type:** 2
- **Project period:** 2020-05-01 → 2028-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10980934

## Citation

> US National Institutes of Health, RePORTER application 10980934, Hemolysis and Free Heme Signaling in Pulmonary Hypertension (2R01HL151447-06). Retrieved via AI Analytics 2026-06-11 from https://api.ai-analytics.org/grant/nih/10980934. Licensed CC0.

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