Wnts are endothelial cell-derived PKD1/PKD2-dependent autocrine/paracrine vasodilators

This study demonstrates that endothelial-derived Wnt9b and Wnt5a act as autocrine/paracrine vasodilators by activating PKD1/PKD2 channels (and Fzd-7 for Wnt5a) to trigger eNOS-mediated signaling, a process initiated by intravascular flow via AT1 receptors that ultimately reduces blood pressure.

The Gist

The Big Picture: A New "Traffic Cop" for Your Blood Vessels

Imagine your body's blood vessels as a massive network of highways. Sometimes, traffic (blood) needs to speed up, and sometimes it needs to slow down to keep the pressure just right. For a long time, scientists knew about the "traffic cops" that tell these highways to widen (vasodilation) or narrow (vasoconstriction).

This paper discovers a brand new type of traffic cop called Wnt proteins. Specifically, the researchers found that two types of Wnts (Wnt9b and Wnt5a) are secreted by the lining of your blood vessels (endothelial cells) to tell the vessels to relax and widen, which lowers your blood pressure.

The Cast of Characters

To understand how this works, let's meet the players in this biological drama:

1. The Endothelial Cells: Think of these as the interior decorators lining the inside of your blood vessel highways. They are the first to feel the rush of blood flowing past them.
2. The Wnt Proteins (Wnt9b & Wnt5a): These are the messenger drones. When the decorators feel the flow of traffic, they launch these drones to send a signal.
3. The PKD1/PKD2 Channels: These are the gates on the endothelial cells. They are like a special door that only opens when the Wnt drones knock on it.
4. The AT1 Receptor: This is the sensor on the decorator's wall. It feels the physical pressure of the blood flowing by.
5. Nitric Oxide (NO): This is the relaxation gas. Once the gates open, the cell releases this gas, which tells the muscle walls of the artery to take a deep breath and relax.

The Story: How It All Works

Here is the step-by-step process described in the paper, using our highway analogy:

1. The Rush Hour (Blood Flow)
When blood flows through your arteries, it creates friction against the inner lining (the endothelial cells). Imagine this like a strong wind blowing against a house.

2. The Sensor Activates (AT1 Receptor)
The endothelial cells have a sensor called the AT1 receptor. When the "wind" (blood flow) hits it, the sensor wakes up. It's like a wind chime that starts ringing when the breeze blows.

3. The Messenger Launch (Wnt Secretion)
Once the sensor rings, it triggers a chain reaction inside the cell. It tells the cell to package up Wnt9b and Wnt5a (the messenger drones) and shoot them out into the bloodstream.

• Note: The researchers found that this process needs a specific enzyme (called Porcupine) to "fatten" the drones so they can fly, and it needs a specific signaling protein (Gq/11) to give the order to launch.

4. The Knock on the Door (PKD1/PKD2 Activation)
These Wnt drones float around and land on the PKD1/PKD2 channels (the gates) on the endothelial cells.

• Wnt9b is a specialist: It only knocks on the PKD1/PKD2 gate.
• Wnt5a is a multitasker: It knocks on the PKD1/PKD2 gate and a backup gate called Fzd-7.

5. The Gate Opens (Calcium Influx)
When the gate opens, it lets a rush of Calcium ions flood into the cell. Think of this as water rushing into a dam to turn on a turbine.

6. The Relaxation Signal (Nitric Oxide)
The rushing calcium turns on the cell's "factory," which produces Nitric Oxide. This gas diffuses out and hits the muscle walls of the artery, telling them to let go of their grip. The artery widens, blood flows easier, and blood pressure drops.

The "What If" Experiments (The Proof)

To prove this theory, the scientists played some "what if" games with mice:

• The "Missing Gate" Mice: They bred mice that were missing the PKD1/PKD2 gates in their blood vessels.
  • Result: When they injected Wnt proteins into these mice, the blood vessels did not relax. The blood pressure didn't drop. This proved that the gates are essential for the Wnt signal to work.
• The "Broken Sensor" Mice: They blocked the AT1 sensor.
  • Result: Even with blood flowing, the cells didn't launch the Wnt drones. No drones meant no relaxation. This proved that the flow sensor starts the whole process.
• The "Hot vs. Cold" Test: They cooled the blood vessels down to stop the cells from working.
  • Result: No Wnt drones were launched. This proved the cells are actively making and sending these proteins, not just releasing them by accident.

Why Does This Matter?

This discovery is a big deal for a few reasons:

1. New Blood Pressure Regulators: We now know that Wnt proteins aren't just for building embryos (which is what we used to think they were for); they are active, circulating chemicals that keep your blood pressure in check every day.
2. The Flow Connection: It explains how your body knows to widen blood vessels when you exercise and blood starts rushing faster. The flow itself triggers the release of these relaxing signals.
3. Disease Clues: If this system breaks down, you might get high blood pressure. Conversely, if it goes haywire (like in sepsis, where blood pressure crashes dangerously low), it might be because too many Wnt drones are being launched, causing the vessels to relax too much.

The Takeaway

Think of your blood vessels as a smart highway system. When traffic (blood flow) picks up, the road lining (endothelial cells) senses the speed, launches a drone (Wnt protein), which knocks on a specific door (PKD1/PKD2), releasing a "relax" gas (Nitric Oxide) that widens the road to prevent a traffic jam (high blood pressure).

This paper identifies the drone, the door, and the sensor, giving us a complete map of a vital biological mechanism we didn't fully understand before.

Technical Summary

1. Problem Statement

While Wingless/Int-1 (Wnt) proteins are well-known regulators of development and cell fate via Frizzled (Fzd) receptors, their physiological role in vascular contractility and blood pressure regulation remains unclear. Specifically:

• It is unknown if Wnts act as circulating vasodilators.
• The functional significance of Wnt binding to Polycystin 1 (PKD1) in native endothelial cells (ECs) is poorly defined.
• The cellular source of vasoactive Wnts and the physiological stimuli (e.g., intravascular flow) that trigger their secretion are not established.
• Previous studies showed flow activates PKD1/PKD2 channels in ECs to cause vasodilation, but the upstream mechanism linking flow to PKD1/PKD2 activation was unknown.

2. Methodology

The authors employed a comprehensive multi-modal approach using in vivo, ex vivo, and in vitro models:

• Genetic Models: Tamoxifen-inducible endothelial cell-specific knockout (ecKO) mice for Pkd1 and Pkd2 (generated by crossing Pkd1fl/fl or Pkd2fl/fl with Cdh5(PAC)-CreERT2 mice).
• Vascular Physiology:
  • Pressurized Myography: Third- and fourth-order mesenteric arteries were cannulated and pressurized to 80 mmHg to measure myogenic tone and diameter changes in response to Wnt9b/Wnt5a, flow, and pharmacological inhibitors.
  • Blood Pressure Measurement: Invasive carotid artery catheterization in anesthetized mice to monitor Mean Arterial Pressure (MAP) during intravenous (i.v.) infusions of Wnt9b, phenylephrine, and prazosin.
• Molecular & Cellular Biology:
  • Electrophysiology: Patch-clamp recordings of non-selective cation currents ($I_{Cat}$) in isolated ECs.
  • Protein Analysis: Western blotting and RT-PCR to quantify PKD1, PKD2, eNOS, Fzd-7, and Wnt proteins in tissues, cells, plasma, and serum.
  • Secretion Assays: ECs and cannulated arterial trees were exposed to static vs. flow conditions (shear stress) at 4°C (inhibits secretion) vs. 37°C to measure Wnt9b/Wnt5a release.
  • Pharmacology: Use of specific inhibitors including:
    • Gd$^{3+}$ (cation channel blocker).
    • L-NNA (eNOS inhibitor) and Apamin (SK channel inhibitor).
    • SRI37892 (Fzd-7 inhibitor), NSC668036 (Dishevelled inhibitor), SP600125 (JNK inhibitor).
    • YM254890 (Gq/11 inhibitor), WntC59 (Porcupine/PORCN inhibitor), Losartan (AT1 receptor blocker).
    • WIF-1 (Wnt inhibitor).
• Nitric Oxide (NO) Assays: Colorimetric detection of nitrite/nitrate in plasma and cell lysates.

3. Key Contributions

This study establishes a novel physiological pathway for blood pressure regulation:

1. Identification of Wnts as Vasodilators: Wnt9b and Wnt5a are identified as circulating factors that induce rapid vasodilation and reduce blood pressure.
2. Mechanism of Action: Wnts activate PKD1/PKD2 channels on endothelial cells, triggering a Ca$^{2+}$-dependent signaling cascade that activates eNOS and small-conductance Ca$^{2+}$-activated K$^{+}$ (SK) channels.
3. Distinct Signaling Pathways:
   • Wnt9b: Acts exclusively via PKD1/PKD2 channels.
   • Wnt5a: Acts via a dual mechanism: PKD1/PKD2 channels and the Frizzled-7 (Fzd-7)/Dishevelled/JNK pathway.
4. Upstream Regulation: Intravascular flow activates Angiotensin II Type 1 (AT1) receptors, which via Gq/11 and Porcupine (PORCN), stimulate the secretion of Wnt9b and Wnt5a from endothelial cells.
5. Autocrine/Paracrine Loop: Flow-induced Wnt secretion creates a local autocrine/paracrine loop that sustains flow-mediated vasodilation (FMV).

4. Key Results

• Wnt-Induced Vasodilation: Intraluminal application of Wnt9b or Wnt5a caused significant vasodilation in wild-type (WT) mesenteric arteries. This effect was abolished in Pkd1 ecKO arteries and significantly reduced in Pkd2 ecKO arteries. Boiled (denatured) Wnts had no effect.
• Blood Pressure Reduction: Intravenous infusion of Wnt9b reduced MAP by 14.6 mmHg in WT mice. In Pkd1 ecKO mice, this reduction was negligible (3.2 mmHg), confirming the dependence on endothelial PKD1.
• Electrophysiology: Wnt9b stimulation increased non-selective cation currents ($I_{Cat}$) in WT ECs, an effect blocked by Gd$^{3+}$ and absent in Pkd1 ecKO cells.
• Downstream Signaling:
  • Wnt9b increased phosphorylated eNOS (p-eNOS) and NO production. This was blocked by Ca$^{2+}$ chelation (BAPTA-AM) and eNOS/SK channel inhibitors.
  • Wnt5a-induced signaling required Fzd-7, Dishevelled, and JNK (blocked by SRI37892, NSC668036, and SP600125, respectively), in addition to PKD1/PKD2.
• Flow-Dependent Secretion:
  • Flow (shear stress) at 37°C increased extracellular Wnt9b and Wnt5a levels; this was absent at 4°C.
  • Secretion was inhibited by Losartan (AT1 blocker), YM254890 (Gq/11 inhibitor), and WntC59 (PORCN inhibitor).
  • Crucially, flow-stimulated secretion was not dependent on EC PKD1 (secretion occurred in Pkd1 ecKO cells), indicating PKD1 is the receptor for Wnts, not the sensor for flow-induced secretion.
  • AT1 receptor knockdown (siRNA) confirmed that both AT1a and AT1b subtypes contribute to flow-induced Wnt secretion.
• In Vivo Validation: WIF-1 (a Wnt inhibitor) reduced flow-mediated dilation in WT arteries to the level seen in Pkd1 ecKO arteries, proving that endogenous Wnts mediate FMV.

5. Significance

• Novel Physiological Pathway: The study redefines the role of Wnt proteins from developmental regulators to acute, circulating vasodilators that maintain vascular tone and lower blood pressure.
• Mechanistic Insight: It resolves the "black box" of how intravascular flow activates PKD1/PKD2 channels, identifying AT1 receptors as the mechanosensor that triggers Wnt secretion, which then activates PKD1/PKD2 in an autocrine/paracrine manner.
• Therapeutic Implications:
  • Hypertension: Dysfunction in this Wnt-PKD1 pathway may contribute to hypertension (as Pkd1 ecKO mice have elevated BP).
  • Sepsis: The authors hypothesize that elevated Wnt5a levels in sepsis patients might drive pathological vasodilation and hypotension via this pathway.
  • Drug Targets: Components like AT1 receptors, PORCN, and specific Wnt isoforms could be targeted to modulate vascular resistance.
• Paradigm Shift: It challenges the view that PKD1 is solely a mechanosensor; here, it acts as a ligand-gated channel for Wnts, while the actual mechanosensing of flow is mediated by AT1 receptors.

In conclusion, this paper elucidates a critical feedback loop where blood flow activates AT1 receptors to secrete Wnts, which then bind to PKD1/PKD2 on endothelial cells to induce nitric oxide-mediated vasodilation, thereby regulating systemic blood pressure.