HTRA1 and aorta stiffening

QuantumTx PEMF technology elicits high temperature requirement A1 protein described in the fat and muscle post. HTRA1 and PEMF looks at come cell culture studies on HTRA1 and other compound secreted by cultured myocytes on cultured breast cancer cells. These studies were published in 2024. This post examines the role of HTRA1 in blood vessel function and postulates that HTRA1 may cause secretion of HTRA1 that promotes compliance of the aorta and other major vessels that tend to stiffen as we age.

HTRA1 in the blood vessels, Song 2024

High temperature requirement A1 (HTRA1) is amember of a family of serine proteases from bacteria to humans. Humans have four isoforms of HTRA, 1-4. The Song review also states that HTRA isoforms might also act as chaperones, i.e. aid in correct protein folding.

Fig 1 HTARA1 cleaves JAG1, a cell surface protein, that binds to a Notch receptor of a second adjacent cell. The In vascular smooth muscle HES and HEY transcription factors repress transcription of genes for proteins like SM22α, α-smooth muscle actin, and smoothelin. The default is the synthetic phenotype. Matrix metalloprotease 9 is increased in this phenotype that not only leads to the degradation of the extracellular matrix but also apoptosis of smooth muscle cells.
Fig2 and associated text discuss the role of HTRA1 in wet age related macular degeneration. FYI, not for this post!

HTRA1 affects the expression of some cytoskeleton proteins in the aorta.

The aorta in an HTRA1 knock out mouse, Ikawati 2018

Often times knocking out a gene makes little difference in the phenotype.

• Fig1 The middle cerebral artery of the knockout mice was surprisingly normal in terms of elastin fibers, alpha smooth muscle actin, and other histological parameters.
• Fig 2 Age of the mouse seemed be be a factor in the geometry of the aorta in the time frame of 16 to 60 weeks, but not in a clear way.
• Fig 3 In culture, HTRA1 knock out smooth muscle cells (VSMC) were more proliferative and invasive.
• Fig 4 PDGF-BB and IGF both increased the migration of HTRA1 knock out vascular smooth muscle cells as well as mm9 activity.
• Fig 5 HTRA1 knockout vascular smooth muscle cells were more prone to H₂O₂ apoptosis, programmed cell death.
• Fig 6  Aortas HtrA1^-/- mice: 5-day-old, SMA, calponin, and vimentin similar 16 and 24 weeks more calponin than WT, but similar SMA, osteopontin, and vimentin. 40 and 52 weeks HtrA1^-/- calponin half WT

• The Media thickness variability was greatest in the HTRA1 knock out mice.
• Elastin and collagen degradation was also greater in the aorta of the knock out mice.
• ⭐Calponin levels in mouse aortas decreased to about half of those in WT mouse aortas at 40 and 52 weeks of age.

Fig 6 and changes in calponin may be a strong observation in the hypothesis that PEMF tuned to release HTRA1 may also mitigate age related aorta stiffening.

Aorta Stiffening and aging, Kajuluri 2021

Stiffness definition, outside the Kajuluri review

Stiffness of an elastic body is the resistance to an applied force. Compliance is the opposite of resistance. The Windkessel vessels, e.g. the aorta, can be mathematically molded to “RC” circuits with resistors and capacitors. The alternating current source is the beating heart. The resistor is the resistance of the circulatory system. The capacitor is the elasticity of the large blood vessels. Flow of electrons is flow of blood. In electrical circuits capacitators cause flow of electrons through the unit has a component that is in phase (resistor) with the AC source as well as out of phase (capacitator) When our major vessels are compliant/elastic, our blood flow is more even with the beating of our hearts.

Measuring stiffness, Kajuluri 2021

High-resolution B-mode ultrasound has been used to measure carotid artery intima-media thickness. Aortic stiffness [measured is measured by carotid femoral pulse wave velocity (CFPWV). Collagen cross linking and elastin degradation were listed as some causes of age related vascular stiffening. This review proposes that 50% of the aorta’s stiffness comes from the cytoskeleton.

The TGFβ connection

The Mogan review cited a Zhu 2018 study that demonstrated age-dependent increases in actin cytoskeletal stiffness have been shown to be positively associated with pro-fibrotic TGFβ expression that also increases integrin receptors that link the cytoskeleton to the extracellular matrix.

If PEMF increases HTRA1 release, could it’s positive effects, if any, be to disable TGFβ rather than it’s minor role in degrading elastin. Never mind that lack of HTRA1 can turn on mm9 in some systems. Let’s take a closer look at the paper cited in the Morgan review.

TGFβ1 and smooth muscle cell stiffness, Zhu 2018

This particular paper is deeply biophysical. Because TGFβ1 is a target for the HTRA1 protease, here we go!

methods

• These authors used a cross between two common strains of rats: Fisher 344 and Brown Norway. F344XBN rats exhibit the same age-related structural changes in the vascular wall that are similar to what his seen in aging humans.
• Pro-fibrotic transforming growth factor β1 (TGFβ1), and thickening of the intima, media and adventitia layers of large vessels is one of these human like changes.
• The aorta of these old and young mice were used as a source of (primary) cultured smooth muscle cells.
• Functionalized magnetic beads were attached to the surface of these cells such that when they twisted in response to a magnetic field, the underlying cytoskeleton was directly impacted.
• . Beads were magnetized with a brief 0.1 mT pulse and twisted in a vertically aligned homogeneous magnetic field (20 Gauss) that was varying sinusoidal in time.
• This sinusoidal twisting magnetic field caused both a rotation and a pivoting displacement of the bead.
• Just as resister in an AC circuit reduces that current that is in phase with the voltage. Recall Ohm’s Law V-IR where I is the current. The capacitor of and RC stores charges and then releases them when the alternating voltage decreases as part of the cycle. This is the “storage” or “out of phase” part of the complex resistance called impedance.
• Many of the elastic elements can be modeled with a Hookean spring. Stored force, F_s = kx, where k is a constant factor such as its stiffness and x is the distance of the deformation, generally small compared to the total possible deformation of the spring.
• The authors defined the ratio of specific applied torque to lateral bead displacements as the complex elastic modulus of the cell, 𝑔^⁎⁡(𝑓) =𝑔 ′(𝑓) +𝑖⁢𝑔 ″(𝑓)
  , where g’ is the storage modulus (cell stiffness), g” is the loss modulus (cell friction), and i² = −1. Yes, this equation involves the square root of -1, which is an imaginary number. Cell stiffness and friction are expressed in units of Pascal per nm (Pa/nm).

The part that is really confusing is stiffness vs elasticity. Stiffness is the ability of an object to resist deformation by an applied force. Elasticity is the ability to return that energy once the force has been removed.

Vinculin and actin introduction

These are some images collected from Wikimedia Commons as well as one of a shock absorber for motor cycles. The energy stored in a depressed shock absorber is returned once the force is removed. With each systole energy is stored in stretched out large blood vessels. During diastole the energy is returned allowing for a more even flow of blood during the cardiac cycle. Zhu 2018 seemed to be focused on actin (think house frame) and vinculin (think joists that join the frame of the house to the roof and floors).

Fig 1 Mechanidal properties of young vs. old VSM cells as measured by MTC

• . (a) Using MTC, we measured cell stiffness (g’, storage modulus) and internal friction (g”, loss modulus) over 5 decades of probing frequency. ..Note that as the mechanical frequency increases, the friction and the stiffness increase. The large pulses of Quantum technology are 15 Hz with microbursts in the 6-7 kHz range. By Faraday Law of induction, what could this mean if moving ions are the mechanical force?
• (b) Hysteresis η (the ratio of g” to g’) detected at 0.75 Hz is not statistically different in young vs old.
• (c) Fetal bovine serum concentration (0.1–10%) was tested on old and young vascular smooth muscle cells. Old VSMC were stiffer regardless of FBS concentration.
• (d) Young vs old VSMC was tested on different values of matrix rigidity. Rigidity is the tendency to maintain shape when an outside force is applied. Rigid surfaces tended to translate into stiffer cells, though stats were not supplied.

Aorta stiffening happens with age.

Fig 2 TGFβ1 in old aorta VSMC

Brief background by way of a Travis and Sheppard 2013 review: Fig 1 TGFβ1 is synthesized as a pro-cytokine with an N-terminal inhibitory region as well as an internal protease called furin. It the latent form is is part of an extracellular matrix associated complex. Fig 2 (Sheppard 2013) Binding of TGFβ1to its receptor results in the phosphorylation of smads, transcription factors that bind to promoters of the collagen gene and related genes.

Returning to the Zhu 2018 study, old aorta VSMC contain 3000 mg of latent TGFβ1 per mg of total protein. Active TGFβ1 was undetectable in young and old aorta VSMC. The latent form of TGFβ1 was also not detectable in young cells. At baseline, the phosphorylation levels of Smad2/3 were also higher in old VSM cells. Addition of exogenous TGFβ1 increased the phosphorylation levels of Smad2/3 in both young and old VSM cells; however, the increase was larger in young VSMCs.

Latent TGFβ1 increases with age.

Fig 3 TGFβ1, actin, and vinculin

Actin is analogous to wood frame of a house. Vinculin is the joists that join the frame to the roof and the floor. Both increase in o VSMC from old aorta vs young. TGFβ1 increases actin expression in VSMC from young mice.

1. Serum and matrix rigidity increased stiffness in aged VSMC
2. Growth factor TGFβ1 signaling through  downstream transcription factor SMAD3 were investigated.
3. Additional TGFβ1 influenced protein levels of filamentous actin and adhesion plaque protein vinculin.
4. The role of TGFβ1 was investigated with pharmaceutical and genetic silencing.
5. TGFβ1 increased cell adherence.
6. Suspecting αβ integrins transmembrane proteins that attach cells to the extracellular matrix, the strain produced by contracting myocytes on collagen gels was investigated. Aged VSMC produced more strain.

Vinculin and actin a molecular spring? This post will not get into the peer reviewed literature supporting this concept.

Fig 4 TGFβ1 induces cell stiffness

Exogenous TGFβ1 stiffens young rat VSMC but not the old VSMC. The young VSMC stiffness increased to a level statistically the same as the old VSMC. This effect does not take into account the latent form. Zhu 2018 used silencing RNA techniques and inhibitors of the TGFβ1 receptor to prove TGFβ1 involvement.

Fig 5 VSMC tethering and TGFβ1

Cells are normally tethered to surfaces via the interactions of integrins with extracellular matrix proteins. Zhu and coauthors had a system with double stranded DNA such that they knew the strength of each molecular tether. Young VSMC adhered better than the old VSMC. TGFβ1 decreased the tethering.

Fig 6 Fourier Transform Traction Microscope

This image describes how FTTM works. FTTM is a convenient way to measure the force a cultured cell exerts on a collagen gel. Old VSMC exerted more

• projected area, old VSMC more spread out
• traction
• maximum cumulative force
• strain energy
• pre-stress force tension force borne by the actin filaments
• amplitude of the contractile moment, a scalar measure of the cell’s contractile strength

Wrapping things up

• HTRA1 affects the expression of some cytoskeleton proteins in the aorta. (Song 2024) The unanswered question is if HTRA1 in the serum can readily diffuse into the vascular smooth muscle. Can PEMF release HTRA1 from vascular smooth muscle as well?
• The smooth muscle cytoskeletal protein calponin is decreased relative to wild type in HTRA1 knockout mice (Ikawati 2018)
• Aorta stiffening increases with age as does latent Latent TGFβ1. Cytoskeletal proteins vinculin and actin seem to be part of the process. Overall, attachment is stronger in old vascular smooth muscle cells. (Zhu 2018)
• Aorta stiffening can be measured (Kajulufi 2021). One could do the experiment the experiment.

References

• Kajuluri LP, Singh K, Morgan KG.(2021) Vascular aging, the vascular cytoskeleton and aortic stiffness. Explor Med. 2021;2:186-197. PEMC free article
• Ikawati M, Kawaichi M, Oka C. (2018) Loss of HtrA1 serine protease induces synthetic modulation of aortic vascular smooth muscle cells. PLoS One. 2018 May 16;13(5):e0196628. PMC free paper
• Song S, Li X, Xue X, Dong W, Li C. (2024) Progress in the Study of the Role and Mechanism of HTRA1 in Diseases Related to Vascular Abnormalities. Int J Gen Med. 2024 Apr 18;17:1479-1491. PMC free paper
• Zhu W, Kim BC, Wang M, Huang J, Isak A, Bexiga NM, Monticone R, Ha T, Lakatta EG, An SS. (2018) TGFβ1 reinforces arterial aging in the vascular smooth muscle cell through a long-range regulation of the cytoskeletal stiffness. Sci Rep. 2018 Feb 8;8(1):2668. PMC free paper