Research into cytokines, and their roles in health and disease, is booming.  This is true across the spectrum of organ systems, including our favorite – skin.  Our work with stem cells as sources of cytokines has driven us to a deeper understanding, and appreciation, for the complexities of cytokines. The database of cytokines, their actions, and interactions, grows daily.  We have begun our own organization schema (cytokine informatics) and wanted share some of that with you.

A Cytokine Network

To reduce complexity in medical knowledge, and to help drives unifying theories, we use models. One really interesting model compares the intercellular communications associated with cytokines to other complex networks. The immune-body cytokine network defines a social architecture of cell interactions examines the density of networks including a social network relationship between students; two different food chain networks; a network of neurons in the cat brain; and the cytokine interface sub-network between immune system cells and non-immune body cells.  Cytokine networks scored highest in density, and took 3 of the top 6 spots. It’s an interesting paper, work a look for you nerdy information junkie types.

OK, now that we know how complex this task is, we can ask your forgiveness in advance if we try to simplify things just a wee bit. We will condense a lot of information down to a little, and we may shave a few fine points. But even so, you are not getting the Time magazine version; or even the Popular Science version. This is more of a graduate level seminar in cell biology of skin and aging.  Bear with us, it’s a starting point, and we can further clarify as needed. Maybe we should offer an online degree program?  🙂

Here is our topical agenda. We will take it in 3 parts.

• Regeneration vs. Repair. We talk about how wound healing relates to regeneration, and yet how it differs, which will ultimately allows us to bring this back around to skin care
• Stages of Healing. Building on the repair paradigm, we look at the classical stages of healing.
• Aging is not Injury – or is it? Now we add the concept of aging, how that affects the processes of healing and regeneration.
• Inflammaging. We focus on the contribution that inflammation makes to aging, and discuss briefly the inflammatory theory of chronic disease and aging as a stepping stone to our discussion of cytokines.
• Cytokines in Healing and Regeneration. Here we begin to explore key cytokines involves with healing, with a special emphasis on those that are inflammatory, and those that are involved with resolving inflammation.
• Inflammatory Healing. What happens to skin when the inflammatory phase just won’t stop. Or we add to it, inadvertently. How to detect the signs of inflammatory healing.
• Fibrotic vs. Fetal Wound Healing. Exploring the key differences between the inflammatory healing associated with older age, and the scarless fetal healing, focusing on several cytokines at the center of this phenomenon.
• Cytokine Therapies. From gene therapy to cell therapy to cytokine pharming to cellular conditioned media, to stem cell orchestras producing cytokine symphonies.  We examine a range of therapeutic approaches to cytokine manipulation. Everything from PICT (therapeutic wounding) to cytokines on call to
• Cytokine Knowledge Base (version alpha). We reveal a knowledge base project we have created specifically to capture known data about cytokines related to skin, healing, anti-aging, and rejuvenation.
• Sample Tables. We are abstracting data from the knowledge base project to create tables showing e.g. what cytokines are responsible for what actions, and when (context); which are inflammatory vs. anti-inflammatory; the differential secretomes of different cell types in culture, including stem cells of differing niches. We will provide commentary on what this means in terms of putative therapeutic effects. You will find some surprising things here. Stay tuned!
• A Cytokinetic View of the Future. Finally, we look down the road 5, 10, and 25 years to show you where this is all going.  Theory, speculation abounds. A fun bonus ride.

PART ONE.

Regeneration vs. Repair

Regenerative medicine is obsessed with wound healing. There are good reasons. The differences, physiologically speaking, between the repair and regeneration of a tissue are subtle.  Healing a wound involves repair to keep the whole organ (e.g. skin) working as designed, but does not guarantee a close copy in terms of functionality or appearance.  If the goal is to re-create the normal defense barrier that skin provides, then a bad looking scar may work as well as a scar-free healing process. Regeneration, on the other hand, restores the tissue functionally,  physiologically, and esthetically – an exact copy, so to speak, of the original.

If we look further down the phylogenetic tree, we find organisms that heal by regeneration throughout their lifespan. Entire new limbs and organs can be grown in response to injury, every bit as functional as the original, and they even look the same. Integument (skin) can be repaired without scarring. But mammals such as ourselves are not quite as adept at this regenerative process. We do not heal wounds this way spontaneously, and yet it seems we once could.  If we reverse ontogenetically, back a few years to when we were mere fetuses at less than 16 weeks gestation, we see this same ability, at least as far as skin is concerned.  Intrauterine (fetal) surgery is quite sophisticated these days; one of the things that has fascinated physicians in this era is the discovery that incisions into fetal skin heal via regeneration, not repair. In other words, without scarring or fibrosis.  Under certain circumstances, we can mimic regeneration in skin wounds, such as by using biodegradable scaffolds (collagen-glycoaminoglycan – the stuff of skin matrix (ecm), and of course a main target in the branch of regenerative medicine concerned with dermal esthetics).

We are now beginning to discover at a biochemical level the essential differences between healing (in adult humans) and regeneration (as in fetuses, and frogs). As we do so, we find ways we can exploit that knowledge to create a true regenerative environment within skin. Why is this important? The current generation of “anti-aging” actives in skin care products are derived from concepts that come mainly from studies of wound healing. There is good reason for this, as you will see in more detail as we explore the stages of wound healing, how it is accomplished, and the outcome.

For example, one key element is the restoration of matrix (collagen and elastin) to “support” a wound (the foundation for the epidermal cells), which is of course one of the things that aging affects dramatically. Most actives are aimed at increasing the generation of matrix.  There is a far more abundant medical literature about wound healing than about wrinkles, but they share many commonalities, especially these architectural dependencies.  Collagen bundle alignment signifies the degree of scarring; normal skin has a basket-weave collagen pattern, while aligned collagen fibers lead to significant scarring.We need to explore the processes that determine how collagen bundles are knitted together. The key factor, it turns out, is stress, mechanical or biochemical.  Mechanical stressors include the presence of edema (excess fluid) or large numbers of immune cells that distort the tissue, and other factors. Chemical stressors are mainly those that promote inflammation, e.g. chemokines that attract white cells to the site of damage (whether mechanical injury or physicochemical e.g. photoaging).

But you might ask at this point … do those matrix  stimulating anti-aging actives, if they are actually working, create a collagen that is just like new,  smooth and pretty, or does it just make whatever is needed to hold new surface cells in place so my skin doesn’t leak or allow bugs and stuff in?  Is it scarring (repair), or scar-free (regenerative)?  That is the pivotal question. And yes, we are going there.

Stages of Healing

Wound healing proceeds via an overlapping pattern of events including

1. Coagulation (not considered a phase by some)
2. Inflammatory
3. Proliferative
4. Matrix and tissue remodeling

The process of repair is mediated in large part by interacting molecular signals, primarily cytokines, that motivate and orchestrate the manifold cellular activities which underscore inflammation and healing (Figure 1).

The initial injury triggers coagulation and an acute local inflammatory response followed by mesenchymal cell recruitment, proliferation and matrix synthesis. Failure to resolve the inflammation can lead to chronic non-healing wounds, whereas uncontrolled matrix accumulation, often involving aberrant cytokine pathways, leads to excess scarring and fibrotic sequelae.

Within minutes post-injury, platelets (thrombocytes) aggregate at the injury site to form a fibrin clot. This clot acts to control active bleeding. The platelets are also themselves sources of key cytokines , including the class known as growth factors (e.g. PDGF).

In the inflammatory phase, various immune system cells are called into action, primarily to deal with debris and any foreign invaders, e.g. bacteria.  Another class of cytokines, called chemokines, primarily attract immune cells.  There are multiple types of white cells involved, and they also are sources of cytokines.  Some cells are phagocytes, which ingest and remove dead cells and debris. There is also migration of cells to the area to begin rebuilding both the foundation (matrix) and keratinocytes.

The proliferative phase is characterized by angiogenesis (new blood vessels), collagen deposition, granulation tissue formation, epithelialization, and wound contraction.  In fibroplasia and granulation tissue formation, fibroblasts grow and form a new, provisional extracellular matrix (ECM) by excreting collagen and fibronectin.Concurrently, re-epithelialization of the epidermis occurs, in which epithelial cells proliferate and ‘crawl’ atop the wound bed, providing cover for the new tissue.

The remodeling phase is marked by wound contraction and maturation. First, the wound is made smaller by the action of myofibroblasts, which establish a grip on the wound edges and contract themselves using a mechanism similar to that in smooth muscle cells. When the myofibroblast’s  roles are close to complete, unneeded cells undergo apoptosis.However, under conditions of inflammation, myofibroblasts may persist, and they contribute to fibrosis and scarring in the skin.  In the maturation and remodeling phase, collagen is remodeled and realigned along tension lines and cells that are no longer needed are removed by apoptosis.  The decision to save or remove a cell is influenced by a number of different cytokines. An imbalance (e.g. excess EGF or “epidermal growth factor” can cause cells that need to be removed to be rescued instead.  This can lead to the piling of new cells upon old ones.

This whole process is not only complex but fragile, and susceptible to interruption or failure leading to the formation of non-healing chronic wounds. Factors which may contribute to this include diabetes, venous or arterial disease, infection, and (you guessed it) aging.

Recall that early in gestation, injured fetal tissues can be completely recreated, without fibrosis, in a process resembling regeneration. One reason why fetuses are not “viable” in those early stages is the immaturity of their immune system (they relies on the mother’s humoral immune system). The key difference between fetal wound healing and adult wound healing is the amount of time spent in the inflammatory phase.  Fetal wounds very rapidly progress from inflammation to proliferative phases.  Thus we can hypothesize that it is the inflammation itself that characterizes adult vs. fetal healing. And there is evidence that the longer inflammation lasts in a skin wound, the more likely the outcome will be scarring & fibrosis.

All of this is mediated by biochemical, principally those called cytokines, including growth factors and chemokines. This ubiquitous class of chemicals act as “signal proteins” to communicate between cells. They provide the language of cells. They are very important to us in our work with mesenchymal stem cells.

Continuing progress in deciphering the essential and complex role of cytokines in wound healing provides opportunities to explore pathways to inhibit/enhance appropriate cytokines to control or modulate pathologic healing, as well as the healing deficiencies that occur with aging. This movement is at the core of anti-aging medicine as applied to dermal aesthetics.

Aging is not Injury – or Is It?

One component of aging is intrinsic, and genetically programmed. Another part is extrinsic, resulting over time from many small injuries. For skin, the commonest cause of damage is related to UV rays. Chemicals of all sorts come into play, both in the environment, and those we ingest. Like death by a thousand paper cuts, these daily events add up. But these small injuries are also somewhat insidious, even sneaky. They may be too small to invoke wound healing mechanisms.

Because blood vessels are not interrupted, platelets are not called into action. In the inflammatory phase of healing, platelet adherence is increased, and the cytokines TGF-beta, TGF-alpha, PDGF are increased. Inflammation occurs, e.g. from oxidative stresses on skin cells, but without tissue disruption there may be blunted cytokine responses to control inflammation once it starts. Decreased nitric oxide, neutrophil diapedesis, and growth factor production is evidenced. Macrophages are less phagocytic. In the proliferative phase, the signals for collagen repletion may be out of balance.  The signals for matrix breakdown via metalloproteinases (MMP’s) may be activated. Aging is associated with higher MMP’s and lower TIMP’s (tissue inhibitors of metalloproteinases). In remodeling, TGF-beta may be released, favoring fibrosis and scar formation within collagen bundles.

So, while aging is not injury, it contributes in a major way to how our bodies respond to the subtle injuries of daily life.  And cytokines, as mediators of inflammation, seem to be at the center of it.

Inflammaging and Anti-inflammaging

Inflammaging refers to a low-grade pro-inflammatory phenotype which accompanies aging in mammals  “A large part of the aging phenotype, including immunosenescence, is explained by an imbalance between inflammatory and anti-inflammatory networks, which results in the low grade chronic pro-inflammatory status we proposed to call inflammaging.” Considering inflammation as a “causative agent in aging” belies the underlying mechanisms whereby the acute inflammatory response is necessary for survival, and efforts to reduce and control the inflammatory response leave the host susceptible to infectious agents and improper healing. The answer is not to eliminate inflammation, but to ensure that it is present only when needed, and that it disappears when not.  A tightly controlled sequence.