Health and Heparan Sulfate Circulation — Connective Tissue is Alive

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Arthritis, Alzheimer’s, diabetes, cardiovascular disease, osteoporosis, cancer, etc. are all diseases of cellular metabolism and secretion.  What goes on inside cells and on their surfaces explains a lot about health and why we get sick.  Cells feed off of what’s around them, use some of those materials to replicate and package up cell-made materials for export.  Eat, replicate and secrete.  Symptoms of disease result if those processes are compromised.

Cell that make Cartilage, Eat Cartilage

The connective tissue that makes up the cartilage of tendons and the non-mineral parts of bones, as well as a layers of skin, is made up of proteins (collagen) and polysaccharides (glycosaminoglycans, GAGs), e.g. heparan sulfate, hyaluronan and chondroitin sulfate, produced by  chondrocytes or fibroblasts.  These proteins and polysaccharides are synthesized and then secreted by cells.  This process goes on continuously, since the connective tissue is alive and literally crawling with cells that make the cartilage.  To keep the connective tissue healthy, the old tissue has to be digested, so that new material can replace it.  Thus, the cells that live in cartilage also eat cartilage.  These cells get all of their nutrients, e.g. protein and carbs, from eating cartilage.  They don’t get glucose and amino acids, or even oxygen (they ferment), from the blood, because there are no blood vessels in cartilage.  The photomicrograph at left shows the red chondrocytes surrounded by a light capsule of heparan sulfate as they burrow through the purple cartilage.  The next micrograph shows the cytoskeleton of actin filaments (stained with a red fluorescent dye, that lies under the cytoplasm of a chondrocyte.  Motor proteins move other proteins, such as syndecans, the proteins to which the heparan sulfate chains are attached, through the  cell membrane (see the animations below.)  The last micrograph shows the green stained microtubule network on which vesicles move to carry heparan sulfate products from one end of the cell to the other (under the actin and past the orange-dyed nucleus) during synthesis and digestion.

Chondrocytes Burrow Through Cartilage

Chondrocytes are the cells that eat and make cartilage, but all of this eating and making goes on at the same time that the cartilage is also holding everything together, i.e. it is still strong.  If cartilage is cut and the cut ends are held tightly together, the chondrocytes will knit the cartilage together and it will become as strong as it was. 

Heparan Sulfate Circulates over the Surface of Cells

Chondrocytes are not actually rigidly embedded in the cartilage, but rather maintain a capsule of heparan sulfate around themselves.  Thus, they continue to secrete a mixture of heparan sulfate, chondroitin sulfate and collagen, but the heparan sulfate is recycled through the capsule and the other molecules merge into the existing cartilage.  Thus, the heparan sulfate is a kind of carrier that keeps the cartilage from “setting up” while it is being made and transported.  Other cells of the body, such as neurons, don’t make cartilage, but they still have heparan sulfate (HS) circulation that is intimately involved in many other processes, such as the action of hormones.  Disruption of HS circulation causes the symptoms of Alzheimer’s or type 1 diabetes, for example, since amyloids assemble as filaments on threads of HS, and the amyloid filaments jam essential HS circulation.  Plaque in atherosclerotic vessels is high in HS content.  HS is also a major component surrounding vessels to form the blood brain barrier and the barrier to protein loss from kidneys into urine or loss into the gut lumin.  Heparin (fragments of HS) is continually released from mast cells in the lining of the gut to prevent pathogens from binding to cell HSPGs. 

HS Sweep the Cell Surface

There is a constant flow of heparan sulfate proteoglycans (HSPGs) through the cell membrane from the rear of the chondrocyte to the front where the HS is digested again and the protein that was embedded in the membrane, syndecan, is recycled to the Golgi for another trip.  HSPGs (animation to left with blue protein and yellow HS) are attached to motor proteins that propel them through the membrane along microfilaments of actin that form the cyctoskeleton just under the membrane in the cortical region of the cell.  Thus, the heparan sulfate of the HSPGs stick out like hair from the cell surface and sweep continuously from the back to the front of the cell.  At the front of the cell, the HS sweeps through the intact cartilage and reverses the process of cartilage assembly.  The chondroitin sulfate, collagen and HSPGs are dragged into the cell and digested.  The protein parts of the HSPGs are transported to the Golgi  and the HS is synthesized along with other cartilage components and moved in vesicles along microtubules before it is secreted.

HS is Secreted at One End and Eaten at the Other

The animation left shows 1) the initial digestion of the cartilage proteins and polysaccharides on the left.  These cartilage components of amino acids and sugars, are used by the chondrocytes as their sole nutrients 2), and to produce new proteoglycans 3) HS and chondroitin sulfate proteoglycans, in the Golgi, are 4) packaged into secretory vesicles and are 5) secreted on the right.  The HS chains, attached to proteins, are 6) swept through the membrane (see the first animation above) toward the front of the cell, leaving the collagen and chondroitin sulfate for form cartilage behind.  In the process, the heparan sulfate proteoglycans 7) disrupt and solublilize old cartilage ahead as the chondrocytes 8) move through the connective tissue like moles digging through soil.

Other Cell Processes Involving Heparan Sulfate:

• Amyloids of Alzheimer’s and type I diabetes assemble bound to HS.
• Hormones bind to receptors wrapped around HS.
• Blood clotting is controlled by HS.
• Complement is controlled by HS.
• Blood brain barrier is composed of HS.
• Kidney protein barrier is composed of HS.
• Inflammation blocks HS synthesis and promotes heparanase synthesis.
• GAGs are animal soluble fiber when eaten and feed gut flora.
• Pathogens bind to HS.
• HIV-TAT is transported between cells by HS circulation.
• Heparin is made by heparanase fragmentation of HSPG in mast cells and is secreted along with histamine. 
• NFkB activation inhibits HSPG production and stimulates heparanase production.
• Heparan sulfate proteoglycans organize nerve synapses and acetylcholine esterase binds to HS. 
• Gastric proteases cleave around heparin binding domains of proteins, e.g. milk, consist of clusters of basic amino acids.  Peptides with heparin binding domain are antimicrobial; all of the heparin binding peptides are subsequently degraded by pancreatic proteases.
• Heparanase is initially secreted inactive and bound to HSPGs, but it remains bound and is internalized again along with the recycling HSPGs, and is activated before being secreted again.
• Allergens and autoantigens are unusual proteins with sequences of three adjacent basic amino acids (arginine or lysine) that require HSPG circulation for presentation of the immune system.  Nuclear proteins that interact with nucleic acids have sequences of four basic amino acids, the nuclear translocation signal, and are therefore common antinuclear auto antigens.

Health Diagrams

Rosacea: Alzheimer’s of the Face

Is Rosacea Caused by Amyloid LL-37, as Alzheimer’s Is Caused by Anti-microbial Abeta?
A recent article in PLoS One (Thanks Daniel!) suggests that the amyloid beta (Abeta) proteins that aggregate to form fibrous plaques in the brain tissue of Alzheimer victims, function as typical defensive anti-microbial peptides (AMPs), similar to the LL-37 cathelicidin implicated in facial tissue in rosacea.  The structural and functional similarities of Abeta and LL-37 suggest to me that Alzheimer’s and rosacea may also be similar in initiation and treatment.  Let’s compare amyloids and AMPs.

[The figure shows a model protein (from ref.) used to examine stain binding to amyloids.  The stains appear to bind to aromatic amino acids spaced evenly between adjacent proteins, but adjacent basic amino acids (blue) are spaced the same way and provide sites for heparin binding.]

Amyloids:

• Amyloid proteins/peptides align into stacks and fibers
• Stacked beta sheets bind amyloid stains: Congo Red, Thioflavin-T
• Fibers form on anionic polymers: heparin, DNA
• Short amyloid stacks are toxic to cells
• Proteases produce multiple sizes of amyloid peptides

Anti-microbial Peptides:

• AMPS typically contain heparin-binding domains -- basic peptides/ plus charge
• Some AMPs, e.g. LL-37, form fibers on DNA, heparin (stain with amyloid stains)
• Toxic to cell membranes
• Kallikrein stimulated by gut flora migrates to face and clips LL-37 to a smaller peptide that binds to host DNA and stimulates the TLR receptor to produce inflammation
• Stomach pepsin hydrolyzes dietary proteins into anti-microbial peptides (heparin is secreted by mast cells onto to the intestinal surface to protect from any amyloid-like effects)
• Defensins, cathelicidins and other AMPs are under transcriptional control of vitamin D receptor

Abeta Is Anti-microbial Like LL-37

Amyloid beta is the well-known source of the fibrous plaques forming brain lesions in Alzheimer’s disease.  The normal function of Abeta has not been firmly established.  The recent article shows data to support Abeta as an anti-microbial peptide comparable to LL-37 against several pathogenic bacteria and yeast.  Knock-out mice deprived of a gene corresponding to Abeta are susceptible to bacterial infections.  The anti-microbial activity present in extracts from Alzheimer’s disease brains was inactivated by anti-Abeta antibodies.

Implications of Abeta as an AMP Like LL-37

The similarities between AMPs and amyloid peptides suggest some implications for both Alzheimer’s disease and rosacea.  Vitamin D is a hormone that binds to a cytoplasmic receptor and the vitD/receptor complex then acts as a transcription factor that controls the expression of defensins in the intestines, LL-37 in facial skin and perhaps Abeta in brains.

Amyloids form fibers on a scaffolding of heparan sulfate (HS).  There is usually an excess of HS on the surface of cells and the HS is rapidly recycled back into cells.  During inflammation, mast cells release heparin, short fragments of HS, that should also inhibit amyloid fiber formation on HS.   Chronic inflammation, however, reduces HS production and may set the stage for amyloid fiber formation.  HS metabolism of the brain may be vitally important to the development of Alzheimer’s disease, especially since the increasing chronic inflammation of aging people should deplete brain HS.

LL-37 forms complexes with DNA from damaged host cells in rosacea skin.  The LL-37/DNA complexes trigger TLRs and inflammation.  LL-37 may normally bind to cell surface HS and chronic inflammation of the skin may cause the shift to pathogenic autoinflammation.  Topical application or perhaps low dose IV heparin may be effective in disrupting the autoinflammation due to LL-37.  Part of the toxicity of LL-37 in the skin may be due to amyloid like structures that could form with inadequate HS and overabundant LL-37 production.  Vitamin D metabolism should also be very important, since LL-37 synthesis is controlled by vitamin D.  This is consistent with the benefits that some rosaceans observe with high doses of vitamin D3 supplements.

references:
Soscia SJ, Kirby JE, Washicosky KJ, Tucker SM, Ingelsson M, Hyman B, Burton MA, Goldstein LE, Duong S, Tanzi RE, Moir RD.  The Alzheimer's disease-associated amyloid beta-protein is an antimicrobial peptide.  PLoS One. 2010 Mar 3;5(3):e9505.

Abedini A, Tracz SM, Cho JH, Raleigh DP.  Characterization of the heparin binding site in the N-terminus of human pro-islet amyloid polypeptide: implications for amyloid formation.  Biochemistry. 2006 Aug 1;45(30):9228-37.

Biancalana M, Makabe K, Koide A, Koide S. Molecular mechanism of thioflavin-T binding to the surface of beta-rich peptide self-assemblies.  J Mol Biol. 2009 Jan 30;385(4):1052-63. Epub 2008 Nov 14.

Heparin, Growth Factors and Rosacea

Knock-out Mice and FGF Receptor Inhibitors Mimic Rosacea
Heparin Nanofibers Loaded with VEGF and FGF Mimic Stem Cells

In previous articles, I have emphasized the mediation of extracellular signaling by heparan sulfate proteoglycans (HSPGs, polysaccharides attached to proteins) and heparin (HS fragments, oligosaccharides) and the sensitivity of HSPG expression and HS degradation by inflammation.  I return to that subject, spurred on by reading two articles that together show both the significance of heparin-mediated growth factors in general and in the specific case of symptom development in rosacea.

FGF Receptor Inhibitors Cause Symptoms Like Rosacea
Fibroblast growth factors stimulate the development of cancers, and antibodies against FGF receptors block cancer growth (see ref.)  FGF receptor inhibiting antibodies are now being used to stop cancers.  Unfortunately,  FGFR antibodies (e.g. cetuximab, panitumumab) also cause symptoms in the skin (telangiectasia, acneiform eruption) similar to the facial inflammation of rosacea.  Similarly, in knock-out mice, that lack the ability to produce FGFR, there are related symptoms.  It appears that lack of some FGF signaling may produce the symptoms of visible blood vessels and pus-filled (though lacking bacteria) follicles of rosacea.

FGF Mediated by HSPG
FGF binds to the heparan sulfate of membrane bound HSPG in pairs and these FGF dimer/heparan sulfate complexes activate a pair of FGF receptors.  The result is activation of protein phosphorylation activity (tyrosine kinase) and normal skin development.  HSPG synthesis is modified by inflammation and heparanase activity is increased.  This suggests that inflammation will decrease FGF signaling and could lead to symptoms of rosacea.

Growth Factors (VEGF, FGF) Bind to Heparin Nanofibers that Mimic Stem Cells
Stem cells produce lots of different growth factors and when stem cells are introduced into damaged cardiovascular tissue, more healing results (see ref.)  To determine if the growth factors produced by the transplanted stem cells was sufficient for the improved healing, fibers made of heparin were dipped into stem cell cultures and the resulting growth factor-coated fibers were injected into damaged tissue.  The heparin-binding growth factors were just as effective at enhancing healing as were the stem cells in previous experiments.  This demonstrated that heparin-binding growth factors were the key to normal repair/revascularization and function.

Rosacea Results from Inflammation and Aberrant Vascularization
Rosacea is poorly understood and is probably numerous diseases that have related symptoms and complex development.  As I indicated in previous articles, neurotransmitters from stimulated facial nerves, enzymes (kallikrein) and cytokines from intestinal interactions with gut flora, mast cell products (heparin, protease) and modified antimicrobial peptides (cathelicidins), as well as cryptic bacteria in facial tissues, may all be involved.  Inflammation in the skin of the face and in the intestines is involved.  Vitamin D, omega-3 fatty acids and anti-oxidants have a variety of responses (sometimes paradoxical) that differ from individual to individual and at different stages in the development of the disease.  Facial inflammation leads to abnormal development of blood vessels (telangiectasia) and in accumulation of lymphocytes and neutrophils (papulopustular rosacea).

Facial Inflammation May Depress HSPG Production and Disrupt FGF Function
One of the key ramifications of persistent facial inflammation may be the depletion of of HSPGs that normally coat cells.  HSPGs are continually produced, reabsorbed and degraded.  The half-life for HSPGs, even those that surround the cells that produce cartilage in connective tissue, is six hours.  HSPGs are also the source of heparin, that is produced as a counter ion bound to histamine and proteases in the secretory granules released by activated mast cells.  Thus, inflammation-based depression of HSPG production, which is also accompanied by heparanase activation, will remove the HSPG coating of cells.  This HSPG coating is needed for normal growth factor function.  Lack of an HSPG matrix on the surface of cells will also result in the migration of growth factors away from where they are normally functional and into adjacent tissue where they may stimulate aberrant development of blood vessels.  This may explain telangiectasia.

Is Topical Heparin a Rosacea Treatment?
Topical heparin does penetrate the skin.  It would appear to be a logical treatment, if HSPG depletion is contributing to symptom development in rosacea.  The length of the heparin fragments may be important.  I am unaware if anyone has tried the heparin lotions that are available for treatment of wounds to minimize scarring, on rosacea.  Heparin may be useful in combination with vitamin D3 and remediation of gut flora in a general scheme to treat rosacea.

refs:
Segaert S, Van Cutsem E.  Clinical signs, pathophysiology and management of skin toxicity during therapy with epidermal growth factor receptor inhibitors.  Ann Oncol. 2005 Sep;16(9):1425-33. Epub 2005 Jul 12.

Webber MJ, Han X, Prasanna Murthy SN, Rajangam K, Stupp SI, Lomasney JW.  Capturing the stem cell paracrine effect using heparin-presenting nanofibres to treat cardiovascular diseases.  J Tissue Eng Regen Med. 2010 Mar 10. [Epub ahead of print]

The Cause of Allegies and Autoimmune Diseases

Keyhole Limpet Hemocyanin (KLH): Internalized Antigen

Scanning the literature for a common protein that can be used as an experimental antigen, it becomes quickly obvious that a favorite is KLH. This would seem to be an odd choice -- why a keyhole limpet protein? But that is the wrong question.

Why is KLH such a good antigen, i.e. why is it readily presented to the host immune system? If you have been reading my posts, you might be thinking about triplets of basic amino acids and that is the answer.

As soon as I remembered the prominent use of KLH as an antigen, I checked the NCBI protein database and immediately found an unusual KKK (triple lysine) near the amino terminus of hemocyanin II ( it comes in two pieces). This triplet explains why KLH is such a good experimental antigen, because it is internalized into antigen presenting cells by its strong heparin-binding domain. Other components, adjuvants, are typically added to the KLH for injection to make sure that a strong local inflammation occurs.

Autoantigens Have Strong Heparin-Binding Triplet

I also learned that Hashimoto’s thyroiditis is an autoimmune disease mediated by the autoantigen thyroid peroxidase. A quick search reveals that thyroid peroxidase is an autoantigen, because it also has a triplet of basic amino acids that can enhance presentation under inflammatory conditions. Grave’s disease of hyperthyroidism is an autoimmune disease in which the thyroid receptor (with a basic triplet) is an autoantigen. The same kind of triplet of basic amino acids was found when I searched today for fire ant antigens and mosquito antigens.

I have also looked for the triplets in protein databases. The triplets are rare in cytoplasmic and extracellular proteins. The proteins that have triplets are usually identified as autoantigens in some disease. The triplets are common in nuclear proteins, since heparin-binding and nucleic acid-binding share the same basic amino acid domains. The nuclear internalization signal also results in rapid cellular internalization, e.g. HIV-TAT, heparanase, IGF-binding proteins. Nuclear proteins are common autoantigens in lupus.

Inflammation Plus Heparin-Binding Internalization: Allergy, Autoimmunity

Chronic inflammation can produce antibodies against proteins (foreign or self) with strong heparin-binding domains (triplets or sometimes neighboring pairs of basic amino acids, lysine or arginine). The generalization explains why particular proteins in pollens, foods, insects, pets, mites, asthma, MS, lupus, celiac, etc. produce antibody responses.