Impaired Capillary Endothelial Cell Differentiation Contributes to pulmonary hypertension in a dynamic Capillary-Alveoli Micro-physiological System and animal models

This study identifies impaired differentiation of capillary endothelial cells driven by CD93-mediated repression of Apelin as a key mechanism in pulmonary hypertension, demonstrating that targeting the Apelin receptor with a biased agonist can restore capillary maturation and improve hemodynamics in both human-derived micro-physiological systems and animal models.

The Gist

The Big Picture: A Clogged Air Highway

Imagine your lungs as a massive, bustling airport. The airways are the runways where planes (oxygen) land, and the blood vessels are the taxiways where the planes move to get their passengers (oxygen) onto the terminal.

In a healthy lung, the taxiways are wide, open, and smooth. But in a disease called Pulmonary Hypertension (PH), these taxiways get clogged, narrowed, and stiff. The pressure builds up, the heart has to work overtime to push blood through, and eventually, the heart fails.

For a long time, doctors thought the problem was just the "big roads" (large arteries). But this study discovered that the real trouble is happening in the tiny side streets (the capillaries) right next to the runways.

The Cast of Characters: The "General" and the "Specialist"

Inside these tiny capillaries, there are two types of workers (cells):

1. The General Workers (gCaps): These are the "construction crew." They are flexible, can repair themselves, and are ready to turn into something else if needed. Think of them as general contractors.
2. The Specialists (aCaps): These are the "air traffic controllers." They are highly specialized cells designed specifically to let oxygen jump from the air into the blood. They are the VIPs of the lung.

The Problem: In a healthy lung, the General Workers naturally grow up to become Specialists. This keeps the system running smoothly.
The Disease: In Pulmonary Hypertension, this growth process gets stuck. The General Workers stay as General Workers and never become Specialists. Because there aren't enough Specialists, the oxygen can't get through, and the whole system breaks down.

The Villain: CD93 (The "Brake Pedal")

The researchers found a specific protein called CD93 that acts like a brake pedal on the General Workers.

• In healthy lungs, the brake is off, so the workers can grow up and become Specialists.
• In sick lungs (PH), the CD93 brake is slammed down hard. It stops the General Workers from turning into Specialists.
• The Result: The "side streets" get dilated (stretched out like a balloon) and blocked, and the oxygen exchange stops working.

The Detective Work: How They Found the Clue

The scientists didn't just guess; they built a mini-lab to solve the mystery:

1. The "City in a Chip" (CAMS): They built a tiny, dynamic model of a lung using human stem cells. It was like a miniature city with roads, buildings, and traffic. When they simulated low oxygen (like being at high altitude), they watched the "General Workers" get stuck and the "Specialists" disappear.
2. The "Fingerprint" (Genetic Sequencing): They looked at the genetic code of lung tissue from sick patients and healthy people. They found that the "Brake Pedal" (CD93) was way too loud in the sick lungs.
3. The "Proof of Concept" (Mice): They took mice with the disease and surgically removed the gene that makes the CD93 brake. Suddenly, the mice got better! The General Workers started turning into Specialists again, and the pressure in their lungs dropped.

The Solution: The "Magic Key" (WN353)

The researchers wanted to fix the problem without just removing the brake (which is hard to do in humans). They looked for a "Magic Key" that could unlock the Specialists.

They knew that a molecule called Apelin usually helps the General Workers turn into Specialists. However, giving Apelin directly is like using a sledgehammer; it fixes the lungs but accidentally breaks the heart (causing heart enlargement).

So, they used a smart, modified version of Apelin called WN353.

• What it does: It acts like a precise key. It unlocks the "Specialist" growth process in the lungs without hitting the heart.
• The Result: In rats with severe lung disease, WN353 didn't just stop the disease; it reversed it. It forced the General Workers to become Specialists, repaired the tiny roads, and lowered the blood pressure—all without hurting the heart.

The Takeaway

This study changes how we see Pulmonary Hypertension. It's not just about stiff arteries; it's about a failure in the lung's ability to build its own oxygen-exchange specialists.

By finding the "Brake" (CD93) and creating a "Smart Key" (WN353) to release it, the researchers have opened a new door for treating this deadly disease. Instead of just trying to force blood through a clogged pipe, we might soon be able to help the lung rebuild its own plumbing.

Technical Summary

1. Problem Statement

Pulmonary Hypertension (PH) is a progressive disease characterized by increased pulmonary arterial pressure, endothelial dysfunction, and right heart failure. While the role of muscularized pulmonary arteries is well-established, the specific alterations in the pulmonary capillary bed (the site of gas exchange) remain poorly understood.

• Key Gap: Recent discoveries identified two distinct capillary endothelial cell types: aerocytes (aCaps), specialized for gas exchange, and general capillary cells (gCaps), which possess stem/progenitor properties. Previous studies suggested a shift in these populations in PH, but the mechanisms driving the loss of aCaps and the failure of gCap-to-aCap differentiation were unclear.
• Challenge: The lack of isolated primary cultures for these specific cell types and the complexity of the alveolar-capillary interface have hindered mechanistic studies.

2. Methodology

The authors employed a multi-modal approach combining human tissue analysis, advanced in vitro modeling, animal models, and molecular biology techniques:

• Human Tissue Analysis:
  • Analyzed lung tissues from PH patients and healthy controls using immunohistochemistry (IHC), immunofluorescence (IF), and 3D tissue clearing.
  • Utilized single-cell RNA sequencing (scRNA-seq) on patient lung samples to identify differentially expressed genes (DEGs) and cell population shifts.
• Capillary-Alveoli Micro-physiological System (CAMS):
  • Developed a novel microfluidic chip integrating human pluripotent stem cell (hPSC)-derived lung epithelial, stromal, and vascular organoids.
  • Used this system to simulate hypoxic conditions and observe dynamic changes in capillary network integrity and barrier function (measured via TEER).
• Genetic Models:
  • Generated BMPR2-mutant hPSCs to model genetic PH.
  • Created a conditional knockout mouse model (Cd34-CreERT2; Cd93-flox/flox) to delete Cd93 specifically in gCaps.
  • Utilized the Sugen 5416/Hypoxia (SuHx) rat model to induce severe PH.
• Molecular Mechanisms:
  • Employed lentiviral overexpression and CRISPR-Cas9 knockdown to manipulate CD93 levels.
  • Used Chromatin Immunoprecipitation (ChIP) assays to verify transcription factor binding.
  • Tested G-protein-biased Apelin Receptor (APLNR) agonists (WN353, WN561) to bypass native Apelin's cardiac side effects.

3. Key Contributions

• Discovery of Capillary Remodeling: Identified that PH is characterized not just by arterial thickening, but by alveolar capillary dilation and a specific failure in the differentiation of gCaps into functional aCaps.
• Identification of CD93 as a Pathogenic Driver: Pinpointed CD93 as a highly upregulated cell-surface receptor on gCaps in PH that acts as a "brake" on differentiation.
• Elucidation of the CD93-SMAD2/3-APLN Axis: Mapped the signaling pathway where CD93 upregulation activates SMAD2/3, which directly binds to the APLN promoter to repress Apelin expression, thereby blocking aCap differentiation.
• Development of CAMS: Established a robust in vitro model (CAMS) that recapitulates the two-phase response of capillaries to hypoxia (initial compensation followed by barrier failure), overcoming previous limitations in studying these cells.
• Therapeutic Innovation: Demonstrated that WN353, a G-protein-biased APLNR agonist, can reverse PH pathology by promoting gCap-to-aCap differentiation without inducing the cardiac hypertrophy associated with native Apelin.

4. Key Results

• Capillary Phenotype in PH:

  • PH patients and SuHx rats showed dilated capillary beds and increased muscularization of capillaries (α-SMA+).
  • There was a significant reduction in aCaps (marked by APLN, EDNRB, HPGD) and an enrichment of gCaps (marked by CD34, APLNR).
  • In BMPR2-mutant hPSCs, differentiation into aCaps was impaired, with persistent high CD34 and low APLN expression.

• Role of CD93:

  • scRNA-seq revealed CD93 as the most significantly upregulated gene in gCaps of PH patients.
  • Conditional Knockout: Deleting Cd93 in Cd34+ cells of SuHx mice significantly reduced right ventricular systolic pressure (RVSP), vascular remodeling, and capillary dilation. It also rescued the population of Apln+ aCaps.
  • Overexpression: Conversely, overexpressing CD93 exacerbated PH phenotypes and reduced aCap numbers.

• Mechanism (CD93 → SMAD2/3 → APLN):

  • CD93 overexpression increased phosphorylated SMAD2/3 (pSMAD2/3) and decreased APLN mRNA/protein.
  • ChIP assays confirmed that SMAD2 binds directly to the APLN promoter.
  • Inhibiting SMAD2/3 (using SB-431542) reversed the suppression of APLN caused by CD93 overexpression.
  • High CD93 levels disrupted endothelial junctions (reduced CDH5, CLDN5) and barrier integrity (lower TEER).

• Therapeutic Efficacy of WN353:

  • Unlike native Apelin, which causes cardiac hypertrophy, the biased agonist WN353 prevented and reversed PH in SuHx rats.
  • WN353 treatment:
    • Significantly reduced RVSP and pulmonary vascular resistance.
    • Reversed capillary dilation and vascular wall thickening.
    • Restored aCap differentiation: Upregulated aCap markers (Ednrb, Car4, Tbx2) and downregulated gCap markers in both in vivo lungs and in vitro hPSC differentiation models.
    • Overcame the suppressive effect of CD93 overexpression on aCap formation.

5. Significance

• Paradigm Shift: This study shifts the focus of PH pathogenesis from purely arterial remodeling to alveolar capillary dysfunction, specifically the failure of progenitor gCaps to mature into functional gas-exchange aCaps.
• Novel Therapeutic Target: It identifies CD93 as a critical upstream regulator and proposes G-protein-biased APLNR agonists (like WN353) as a superior therapeutic strategy. This approach targets the endothelial defect directly while avoiding the cardiotoxicity that has historically limited Apelin-based therapies.
• Model Advancement: The CAMS model provides a critical tool for studying the dynamic interplay between hypoxia, capillary integrity, and cell differentiation in human-relevant systems, bridging the gap between in vitro cell culture and in vivo physiology.

In conclusion, the paper establishes that impaired gCap-to-aCap differentiation driven by the CD93-SMAD2/3-APLN axis is a central mechanism in PH, and that restoring this differentiation via biased APLNR agonists offers a promising new avenue for treating pulmonary vascular disease.